Antibodies reactive with B7-H3 and uses thereof

ABSTRACT

The present invention relates to antibodies that are immunoreactive to the mammalian, and more particularly, the human B7-H3 receptor and to uses thereof, particularly in the treatment of cancer and inflammation. The invention thus particularly concerns humanized B7-H3-reactive antibodies that are capable of mediating, and more preferably enhancing the activation of the immune system against cancer cells that are associated with a variety of human cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/331,876 (filed Jul. 15, 2014; pending), which application is adivisional of U.S. patent application Ser. No. 13/463,924 (filed May 4,2012; now U.S. Pat. No. 8,802,091), which application claims priority toU.S. Patent Application No. 61/482,771 (filed May 5, 2011), and is acontinuation-in-part of PCT/US2011/026689 (filed Mar. 1, 2011; expired),which application claims priority to U.S. Patent Application Ser. No.61/311,057 (filed Mar. 5, 2010), 61/310,695 (filed Mar. 4, 2010) and61/310,692 (filed Mar. 4, 2010), each of which applications is hereinincorporated by reference in its entirety and to which priority isclaimed.

REFERENCE TO SEQUENCE LISTING

This application includes one or more Sequence Listings pursuant to 37C.F.R. 1.821 et seq., which are disclosed in computer-readable media(filed name: 1301-0073C-ST25, created May 31, 2016, and having a size of104,070 bytes), which file is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to antibodies that are immunoreactive tothe mammalian, and more particularly, the human B7-H3 receptor and touses thereof, particularly in the treatment of cancer and inflammation.The invention thus particularly concerns humanized B7-H3-reactiveantibodies that are capable of mediating, and more preferably enhancingthe activation of the immune system against cancer cells that areassociated with a variety of human cancers.

Description of Related Art

The growth and metastasis of tumors depends to a large extent on theircapacity to evade host immune surveillance and overcome host defenses.Most tumors express antigens that can be recognized to a variable extentby the host immune system, but in many cases, an inadequate immuneresponse is elicited because of the ineffective activation of effector Tcells (Khawli, L. A. et al. (2008) “Cytokine, Chemokine, andCo-Stimulatory Fusion Proteins for the Immunotherapy of Solid Tumors,”Exper. Pharmacol. 181:291-328).

CD4+ T-lymphocytes are the essential organizers of most mammalian immuneand autoimmune responses (Dong, C. et al. (2003) “Immune Regulation byNovel Costimulatory Molecules,” Immunolog. Res. 28(1):39-48). Theactivation of CD4+ helper T-cells has been found to be mediated throughco-stimulatory interactions between Antigen Presenting Cells and naiveCD4+T-lymphocytes. Two interactions are required (Viglietta, V. et al.(2007) “Modulating Co-Stimulation,” Neurotherapeutics 4:666-675; Korman,A. J. et al. (2007) “Checkpoint Blockade in Cancer Immunotherapy,” Adv.Immunol. 90:297-339). In the first interaction, an Antigen PresentingCell must display the relevant target antigen bound to the cell's majorhistocompatibility complex so that it can bind to the T-cell Receptor(“TCR”) of a naive CD4+ T-lymphocyte. In the second interaction, aligand of the Antigen Presenting Cell must bind to a CD28 receptor ofthe CD4+ T-lymphocyte (Dong, C. et al. (2003) “Immune Regulation byNovel Costimulatory Molecules,” Immunolog. Res. 28(1):39-48; Lindley, P.S. et al. (2009) “The Clinical Utility Of Inhibiting CD28-MediatedCostimulation,” Immunol. Rev. 229:307-321). CD4+ helper T-cellsexperiencing both stimulatory signals are then capable of responding tocytokines (such as Interleukin-2 and Interleukin-12 to develop into Th1cells. Such cells produce interferon-gamma (IFN-γ) and tumor necrosisfactor-alpha (TNF-α), which mediate inflammatory responses to targetcells expressing the target antigen. B-cell activation and proliferationalso occurs, resulting in antibody production specific for the targetantigen (Bernard, A. et al. (2005) “T and B Cell Cooperation: A Dance ofLife and Death,” Transplantation 79:S8-S11). In the absence of bothco-stimulatory signals during TCR engagement, T cells enter afunctionally unresponsive state, referred to as clonal anergy (Khawli,L. A. et al. (2008) “Cytokine, Chemokine, and Co-Stimulatory FusionProteins for the Immunotherapy of Solid Tumors,” Exper. Pharmacol.181:291-328). In pathologic states, Th1 cells are the key players ofvarious organ-specific autoimmune diseases, such as type I diabetes,rheumatoid arthritis, and multiple sclerosis (Dong, C. et al. (2003)“Immune Regulation by Novel Costimulatory Molecules,” Immunolog. Res.28(1):39-48).

I. The B7 Superfamily and B7-H3

Investigations into the ligands of the CD28 receptor have led to thecharacterization of a set of related molecules known as the B7Superfamily (Coyle, A. J. et al. (2001) “The Expanding B7 Superfamily:Increasing Complexity In Costimulatory Signals Regulating T CellFunction,” Nature Immunol. 2(3):203-209; Sharpe, A. H. et al. (2002)“The B7-CD28 Superfamily,” Nature Rev. Immunol. 2:116-126; Greenwald, R.J. et al. (2005) “The B7 Family Revisited,” Ann. Rev. Immunol.23:515-548; Collins, M. et al. (2005) “The B7 Family OfImmune-Regulatory Ligands,” Genome Biol. 6:223.1-223.7; Loke, P. et al.(2004) “Emerging Mechanisms Of Immune Regulation: The Extended B7 FamilyAnd Regulatory T Cells.” Arthritis Res. Ther. 6:208-214; Korman, A. J.et al. (2007) “Checkpoint Blockade in Cancer Immunotherapy,” Adv.Immunol. 90:297-339; Flies, D. B. et al. (2007) “The New B7s: Playing aPivotal Role in Tumor Immunity,” J. Immunother. 30(3):251-260; Agarwal,A. et al. (2008) “The Role Of Positive Costimulatory Molecules InTransplantation And Tolerance,” Curr. Opin. Organ Transplant.13:366-372; Lenschow, D. J. et al. (1996) “CD28/B7 System of T CellCostimulation,” Ann. Rev. Immunol. 14:233-258; Wang, S. et al. (2004)“Co-Signaling Molecules Of The B7-CD28 Family In Positive And NegativeRegulation Of T Lymphocyte Responses,” Microbes Infect. 6:759-766).There are currently seven known members of the family: B7.1 (CD80), B7.2(CD86), the inducible co-stimulator ligand (ICOS-L), the programmeddeath-1 ligand (PD-L1), the programmed death-2 ligand (PD-L2), B7-H3 andB7-H4 (Collins, M. et al. (2005) “The B7 Family Of Immune-RegulatoryLigands,” Genome Biol. 6:223.1-223.7).

B7 family members are immunoglobulin superfamily members with animmunoglobulin-V-like and an immunoglobulin-C-like domain (e.g.,IgV-IgC) (Sharpe, A. H. et al. (2002) “The B7-CD28 Superfamily,” NatureRev. Immunol. 2:116-126). The IgV and IgC domains of B7-family membersare each encoded by single exons, with additional exons encoding leadersequences, transmembrane and cytoplasmic domains. The cytoplasmicdomains are short, ranging in length from 19 to 62 amino-acid residuesand can be encoded by multiple exons (Collins, M. et al. (2005) “The B7Family Of Immune-Regulatory Ligands,” Genome Biol. 6:223.1-223.7). B7-H3is unique in that the major human form contains two extracellular tandemIgV-IgC domains (i.e., IgV-IgC-IgV-IgC) (Collins, M. et al. (2005) “TheB7 Family Of Immune-Regulatory Ligands,” Genome Biol. 6:223.1-223.7).Members of the B7 family are predicted to form back-to-back,non-covalent homodimers at the cell surface, and such dimers have beenfound with respect to B7-1 (CD80) and B7-2 (CD86).

B7-1 (CD80) and B7-2 (CD86) exhibit have dual specificity for thestimulatory CD28 receptor and the inhibitory CTLA-4 (CD152) receptor(Sharpe, A. H. et al. (2002) “The B7-CD28 Superfamily,” Nature Rev.Immunol. 2:116-126).

Although initially thought to comprise only 2 Ig domains (IgV-IgC)(Chapoval, A. et al. (2001) “B7-H3: A Costimulatory Molecule For T CellActivation and IFN-γ Production,” Nature Immunol. 2:269-274; Sun, M. etal. (2002) “Characterization of Mouse and Human B7-H3 Genes,” J.Immunol. 168:6294-6297) a four immunoglobulin extracellular domainvariant (“4Ig-B7-H3”) has been identified and found to be more commonhuman form of the protein (Sharpe, A. H. et al. (2002) “The B7-CD28Superfamily,” Nature Rev. Immunol. 2:116-126). No functional differencehas been observed between these two forms, since the natural murine form(2Ig) and the human 4Ig form exhibit similar function (Hofmeyer, K. etal. (2008) “The Contrasting Role Of B7-H3,” Proc. Natl. Acad. Sci.(U.S.A.) 105(30):10277-10278). The 4Ig-B7-H3 molecule inhibits thenatural killer cell-mediated lysis of cancer cells (Castriconi, R. etal. “Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated MoleculeThat Exerts A Protective Role From An NK Cell-Mediated Lysis,” Proc.Natl. Acad. Sci. (U.S.A.) 101(34): 12640-12645). The human B7-H3 (2Igform) has been found to promote T-cell activation and IFN-γ productionby binding to a putative receptor on activated T cells (Chapoval, A. etal. (2001) “B7-H3: A Costimulatory Molecule For T Cell Activation andIFN-γ Production,” Nature Immunol. 2:269-274; Xu, H. et al. (2009)“MicroRNA miR-29 Modulates Expression of Immunoinhibitory MoleculeB7-H3: Potential Implications for Immune Based Therapy of Human SolidTumors,” Cancer Res. 69(15):5275-6281). Both B7-H4 and B7-H1 are potentinhibitors of immune function when expressed on tumor cells (Flies, D.B. et al. (2007) “The New B7s: Playing a Pivotal Role in TumorImmunity,” J. Immunother. 30(3):251-260).

The mode of action of B7-H3 is complex, as the protein mediates both Tcell co-stimulation and co-inhibition (Hofmeyer, K. et al. (2008) “TheContrasting Role Of B7-H3,” Proc. Natl. Acad. Sci. (U.S.A.)105(30):10277-10278; Martin-Orozco, N. et al. (2007) “InhibitoryCostimulation And Anti-Tumor Immunity,” Semin Cancer Biol.17(4):288-298; Subudhi, S. K. et al. (2005) “The Balance Of ImmuneResponses: Costimulation Verse Coinhibition,” J. Mol. Med. 83:193-202).B7-H3 binds to (TREM)-like transcript 2 (TLT-2) and co-stimulates T cellactivation, but also binds to as yet unidentified receptor(s) to mediateco-inhibition of T cells. In addition, B7-H3, through interactions withunknown receptor(s) is an inhibitor for natural killer cells andosteoblastic cells (Hofmeyer, K. et al. (2008) “The Contrasting Role OfB7-H3,” Proc. Natl. Acad. Sci. (U.S.A.) 105(30):10277-10278). Theinhibition may operate through interactions with members of the majorsignaling pathways through which T cell receptor (TCR) regulates genetranscription (e.g., NFTA, NF-κB, or AP-1 factors).

B7-H3 co-stimulates CD4+ and CD8+ T-cell proliferation. B7-H3 alsostimulates IFN-γ production and CD8+ lytic activity (Chapoval, A. et al.(2001) “B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-γProduction,” Nature Immunol. 2:269-274; Sharpe, A. H. et al. (2002) “TheB7-CD28 Superfamily,” Nature Rev. Immunol. 2:116-126). However, theprotein also possibly acts through NFAT (nuclear factor for activated Tcells), NF-κB (nuclear factor kappa B), and AP-1 (Activator Protein-1)factors to inhibit T-cell activation (Yi. K. H. et al. (2009) “FineTuning The Immune Response Through B7-H3 And B7-H4,” Immunol. Rev.229:145-151). B7-H3 is also believed to inhibit Th1, Th2, or Th17 invivo (Prasad, D. V. et al. (2004) “Murine B7-H3 Is A Negative RegulatorOf T Cells,” J. Immunol. 173:2500-2506; Fukushima, A. et al. (2007)“B7-H3 Regulates The Development Of Experimental Allergic ConjunctivitisIn Mice,” Immunol. Lett. 113:52-57; Yi. K. H. et al. (2009) “Fine TuningThe Immune Response Through B7-H3 And B7-H4,” Immunol. Rev.229:145-151). Several independent studies have shown that humanmalignant tumor cells exhibit a marked increase in expression of B7-H3protein and that this increased expression was associated with increaseddisease severity (Zang, X. et al. (2007) “The B7 Family And CancerTherapy: Costimulation And Coinhibition,” Clin. Cancer Res.13:5271-5279), suggesting that B7-H3 is exploited by tumors as an immuneevasion pathway (Hofmeyer, K. et al. (2008) “The Contrasting Role OfB7-H3,” Proc. Natl. Acad. Sci. (U.S.A.) 105(30):10277-10278).

Molecules that block the ability of a B7 molecule to bind to a T-cellreceptor (e.g., CD28) inhibit the immune system and have been proposedas treatments for autoimmune disease (Linsley, P. S. et al. (2009) “TheClinical Utility Of Inhibiting CD28-Mediated Co-Stimulation,” Immunolog.Rev. 229:307-321). Neuroblastoma cells expressing 4Ig-B7-H3 treated withanti-4Ig-B7-H3 antibodies were more susceptible to NK cells. However, itis unclear whether this activity can be attributed to only antibodiesagainst the 4Ig-B7-H3 form because all reported antibodies raisedagainst the 4Ig-B7-H3 also bound the two Ig-like form of B7H3(Steinberger, P. et al. (2004) “Molecular Characterization of Human4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains,” J.Immunol. 172(4): 2352-2359 and Castriconi et al. (2004) “IdentificationOf 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts AProtective Role From An NK Cell-Mediated Lysis,” Proc. Natl. Acad. Sci.(U.S.A.) 101(34):12640-12645).

B7-H3 is not expressed on resting B or T cells, monocytes, or dendriticcells, but it is induced on dendritic cells by IFN-γ and on monocytes byGM-CSF (Sharpe, A. H. et al. (2002) “The B7-CD28 Superfamily,” NatureRev. Immunol. 2:116-126). The receptor(s) that bind B7-H3 have not beenfully characterized. Early work suggested one such receptor would needto be rapidly and transiently up-regulated on T cells after activation(Loke, P. et al. (2004) “Emerging Mechanisms Of Immune Regulation: TheExtended B7 Family And Regulatory T Cells.” Arthritis Res. Ther.6:208-214). Recently, the (TREM)-like transcript 2 (TLT-2, or TREML2)receptor (King, R. G. et al. (2006) “Trem-Like Transcript 2 Is ExpressedOn Cells Of The Myeloid/Granuloid And B Lymphoid Lineage And IsUp-Regulated In Response To Inflammation,” J. Immunol. 176:6012-6021;Klesney-Tait, J. et al. (2006) “The TREM Receptor Family And SignalIntegration,” Nat. Immunol. 7:1266-1273; Yi. K. H. et al. (2009) “FineTuning The Immune Response Through B7-H3 And B7-H4,” Immunol. Rev.229:145-151), which is expressed on myeloid cells has been shown to becapable of binding B7-H3, and of thereby co-stimulating the activationof CD8+ T cells in particular (Zang, X. et al. (2003) “B7x: A WidelyExpressed B7 Family Member That Inhibits T Cell Activation,” Proc. Natl.Acad. Sci. (U.S.A.) 100:10388-10392; Hashiguchi, M. et al. (2008)“Triggering Receptor Expressed On Myeloid Cell-Like Transcript 2 (TLT-2)Is A Counter-Receptor For B7-H3 And Enhances T Cell Responses,” Proc.Natl. Acad. Sci. (U.S.A.) 105(30):10495-10500; Hofmeyer, K. et al.(2008) “The Contrasting Role Of B7-H3,” Proc. Natl. Acad. Sci. (U.S.A.)105(30): 10277-10278).

In addition to its expression on neuroblastoma cells, human B7-H3 isalso known to be expressed on a variety of other cancer cells (e.g.,gastric, ovarian and non-small cell lung cancers). B7-H3 proteinexpression has been immunohistologically detected in tumor cell lines(Chapoval, A. et al. (2001) “B7-H3: A Costimulatory Molecule For T CellActivation and IFN-γ Production,” Nature Immunol. 2:269-274; Saatian, B.et al. (2004) “Expression Of Genes For B7-H3 And Other T Cell Ligands ByNasal Epithelial Cells During Differentiation And Activation,” Amer. J.Physiol. Lung Cell. Mol. Physiol. 287:L217-L225; Castriconi et al.(2004) “Identification Of 4Ig-B7-H3 As A Neuroblastoma-AssociatedMolecule That Exerts A Protective Role From An NK Cell-Mediated Lysis,”Proc. Natl. Acad. Sci. (U.S.A.) 101(34):12640-12645); Sun, M. et al.(2002) “Characterization of Mouse and Human B7-H3 Genes,” J. Immunol.168:6294-6297). mRNA expression has been found in heart, kidney, testes,lung, liver, pancreas, prostate, colon, and osteoblast cells (Collins,M. et al. (2005) “The B7 Family Of Immune-Regulatory Ligands,” GenomeBiol. 6:223.1-223.7). At the protein level, B7-H3 is found in humanliver, lung, bladder, testis, prostate, breast, placenta, and lymphoidorgans (Hofmeyer, K. et al. (2008) “The Contrasting Role Of B7-H3,”Proc. Natl. Acad. Sci. (U.S.A.) 105(30):10277-10278).

II. Therapeutic Antibodies

In addition to their known uses in diagnostics, antibodies have beenshown to be useful as therapeutic agents. For example, immunotherapy, orthe use of antibodies for therapeutic purposes has been used in recentyears to treat cancer. Passive immunotherapy involves the use ofmonoclonal antibodies in cancer treatments (see for example, DEVITA,HELLMAN, AND ROSENBERG'S CANCER: PRINCIPLES & PRACTICE OF ONCOLOGY,EIGHTH EDITION (2008), DeVita, V. et al. Eds., Lippincott Williams &Wilkins, Philadelphia, Pa., pp. 537-547, 2979-2990). These antibodiescan have inherent therapeutic biological activity both by directinhibition of tumor cell growth or survival and by their ability torecruit the natural cell killing activity of the body's immune system.These agents can be administered alone or in conjunction with radiationor chemotherapeutic agents. Rituximab and Trastuzumab, approved fortreatment of non-Hodgkin's lymphoma and breast cancer, respectively, areexamples of such therapeutics. Alternatively, antibodies can be used tomake antibody conjugates in which the antibody is linked to a toxicagent and directs that agent to the tumor by specifically binding to thetumor. Gemtuzumab ozogamicin is an example of an approved antibodyconjugate used for the treatment of leukemia.

Monoclonal antibodies that bind to cancer cells and have potential usesfor diagnosis and therapy have been disclosed (see, for example, thefollowing patent applications which disclose, inter alia, some molecularweights of target proteins: U.S. Pat. No. 6,054,561 (200 kD c-erbB-2(Her2), and other unknown antigens 40-200 KD in size) and U.S. Pat. No.5,656,444 (50 kD and 55 kD oncofetal protein)). Examples of antibodiesin clinical trials and/or approved for treatment of solid tumorsinclude: Trastuzumab (antigen: 180 kD, HER2/neu), Edrecolomab (antigen:40-50 kD, Ep-CAM), Anti-human milk fat globules (HMFG1) (antigen &gt;200kD, HMW Mucin), Cetuximab (antigens: 150 kD and 170 kD, EGF receptor),Alemtuzumab (antigen: 21-28 kD, CD52), and Rituximab (antigen: 35 kD,CD20).

The antigen targets of trastuzumab (Her-2 receptor), which is used totreat breast cancer, and cetuximab (EGF receptor), which is in clinicaltrials for the treatment of several cancers, are present at somedetectable level on a large number of normal human adult tissuesincluding skin, colon, lung, ovary, liver, and pancreas. The margin ofsafety in using these therapeutics is possibly provided by thedifference in the levels of antigen expression or in access of oractivity of the antibody at these sites.

Another type of immunotherapy is active immunotherapy, or vaccination,with an antigen present on a specific cancer(s) or a DNA construct thatdirects the expression of the antigen, which then evokes the immuneresponse in the individual, i.e., to induce the individual to activelyproduce antibodies against their own cancer. Active immunization has notbeen used as often as passive immunotherapy or immunotoxins.

Several models of disease (including cancer) progression have beensuggested. Theories range from causation by a singleinfective/transforming event to the evolution of an increasingly“disease-like” or “cancer-like” tissue type leading ultimately to onewith fully pathogenic or malignant capability. Some argue that withcancer, for example, a single mutational event is sufficient to causemalignancy, while others argue that subsequent alterations are alsonecessary. Some others have suggested that increasing mutational loadand tumor grade are necessary for both initiation as well as progressionof neoplasia via a continuum of mutation-selection events at thecellular level. Some cancer targets are found only in tumor tissues,while others are present in normal tissues and are up regulated and/orover-expressed in tumor tissues. In such situations, some researchershave suggested that the over-expression is linked to the acquisition ofmalignancy, while others suggest that the over-expression is merely amarker of a trend along a path to an increasing disease state.

In some cases, cancer targets, such as oncoproteins expressed orover-expressed in tumors, have been shown to be present during embryonicand fetal development and serve as a regulator of growth anddifferentiation. Some researchers have found that the expression ofthese oncoproteins during embryonic and fetal development appear to berestricted to specific tissues and also restricted to specific stages ofdevelopment. In contrast, the expression of these oncoproteins in theadult has been shown to be associated with over-expression in tumorgrowth and/or a malfunction of tumor suppressor proteins.

An ideal diagnostic and/or therapeutic antibody would be specific for anantigen present on a large number of cancers, but absent or present onlyat low levels on any normal tissue. The discovery, characterization, andisolation of a novel antibody capable of binding to an antigen that isspecifically associated with cancer(s) would be useful in many ways.First, the antibody would have biological activity against such cancercells and be able to recruit the immune system's response to therebytreat the disease. The antibody could be administered as a therapeuticalone or in combination with current treatments or used to prepareimmunoconjugates linked to toxic agents. An antibody with the samespecificity but with low or no biological activity when administeredalone could also be useful in that an antibody could be used to preparean immunoconjugate with a radioisotope, a toxin, or a chemotherapeuticagent or liposome containing a chemotherapeutic agent, with theconjugated form being biologically active by virtue of the antibodydirecting the toxin to the antigen-containing cells.

As discussed above, antibodies and other molecules that thatspecifically bind to B7-H3 have been described (see, U.S. Pat. Nos.7,527,969; 7,368,554; 7,358,354; and 7,279,567; United States PatentApplication Publications Nos. US 20090087416; US 20090022747; US20090018315; US2008116219; US20080081346; US 20050202536; US20030103963;US20020168762; PCT Publications Nos. WO 2008/116219; WO 2006/016276; WO2004/093894; WO 04/001381; WO 2002/32375; WO 2002/10187 and WO2001/094413; EP 1292619B; Modak, S. et al. (March 1999)“Disialoganglioside GD2 And Antigen 8H9: Potential Targets ForAntibody-Based Immunotherapy Against Desmoplastic Small Round Cell Tumor(DSRCT) And Rhabdomyosarcoma (RMS),” Proceedings Of The AmericanAssociation For Cancer Research Annual Meeting, Vol. 40:474 (90^(th)Annual Meeting Of The American Association For Cancer Research;Philadelphia, Pa., US; Apr. 10-14, 1999; Modak, S. et al. (March 2000)“Radioimmunotargeting To Human Rhabdomyosarcoma Using MonoclonalAntibody 8H9,” Proc. Am. Assoc. Cancer Res. 41:724; Modak, S. et al.(2001) “Monoclonal Antibody 8H9 Targets A Novel Cell Surface AntigenExpressed By A Wide Spectrum Of Human Solid Tumors,” Cancer Res.61(10):4048-4054; Steinberger, P. et al. (2004) “MolecularCharacterization of Human 4Ig-B7-H3, a Member of the B7 Family with FourIg-Like Domains,” J. Immunol. 172(4):2352-2359; Xu, H. et al. (2009)“MicroRNA miR-29 Modulates Expression of Immunoinhibitory MoleculeB7-H3: Potential Implications for Immune Based Therapy of Human SolidTumors,” Cancer Res. 69(15):5275-6281).

Nevertheless, one aspect desirable for an ideal diagnostic and/ortherapeutic antibody would be the discovery and characterization ofnovel antibodies capable of mediating, and particularly of enhancing theactivation of the immune system against cancer cells (especially humancancer cells) that are associated with a variety of cancers. Suchcompositions would also be useful for drug discovery (e.g., smallmolecules) and for further characterization of cellular regulation,growth, and differentiation.

Thus, despite all prior advances, a need remains for improvedcompositions capable of binding to cancer cells and of facilitating ormediating an immune response against cancer cells. Such compositions maybe used to diagnose and treat such cancers. There exists a further need,based on the discoveries disclosed herein, for novel compositions thatspecifically recognize dual targets on the surface of cells, and whichcan thereby modulate, either by reducing or enhancing, the capabilitiesof B7-H3 to mediate T-cell activation or by recognizing and killingcancer cells that express B7-H3. It is an object of this invention toidentify such compositions. It is another object to provide novelcompounds for use in the assay of B7-H3 expression.

As described in detail below, the present invention relates to novelantibodies, including in particular dual affinity retargeting reagents(“DARTS”) that comprise modulators of B7-H3 T-cell activation, that arecapable of influencing T-cell activation as well as novel antibodiesthat bind to B7-H3 receptors of cancer cells and facilitate or mediatethe death of such cells. The present invention is directed to suchcompositions and to their uses in diagnostics and in the treatment ofdiseases such as cancer.

SUMMARY OF THE INVENTION

The present invention relates to antibodies that are immunoreactive tothe mammalian, and more particularly, the human B7-H3 receptor and touses thereof, particularly in the treatment of cancer and inflammation.The invention thus particularly concerns humanized B7-H3-reactiveantibodies that are capable of mediating, and more preferably enhancingthe activation of the immune system against cancer cells that areassociated with a variety of human cancers.

In detail, the invention provides an isolated antibody, wherein theisolated antibody comprises an antibody light chain having the sequenceSEQ ID NO: 117 or an antibody heavy chain having the sequence SEQ ID NO:119. The invention particularly concerns the embodiments of suchantibody wherein the isolated antibody comprises the antibody lightchain having the sequence SEQ ID NO: 117; wherein the isolated antibodycomprises the antibody heavy chain having the sequence SEQ ID NO: 119;and wherein the isolated antibody is an antibody whose light chains eachhave the sequence SEQ ID NO: 117 and whose heavy chain has the sequenceSEQ ID NO: 119.

The invention particularly concerns the embodiments of such antibodieswherein the antibody binds to B7-H3 that is endogenously expressed onthe surface of a cancer cell and/or wherein the antibody binds to B7-H3expressed on the surface of a cancer cell and is internalized into thecell upon binding to the B7-H3.

The invention further provides a nucleic acid molecule that encodes anyof the above-described isolated antibodies. The invention particularlyconcerns the embodiments of such nucleic acid molecule wherein thenucleic acid molecule has the light chain sequence SEQ ID NO: 118;wherein the nucleic acid molecule has the heavy chain sequence SEQ IDNO: 120; or wherein the nucleic acid molecule has both the light chainsequence SEQ ID NO: 118 and the heavy chain sequence SEQ ID NO: 120.

The invention further provides a vector comprising any of theabove-described nucleic acid molecules. The invention further provides ahost cell comprising any such vector. The invention further provides acell that expresses any of the above-described antibodies.

The invention further provides a pharmaceutical composition comprising:(i) a therapeutically effective amount of any of the above-describedisolated antibodies; and (ii) a pharmaceutically acceptable carrier. Theinvention particularly concerns the embodiments of such pharmaceuticalcomposition, in which the pharmaceutical composition further comprisesone or more additional anti-cancer agents (such as a chemotherapeuticagent, a radiation therapeutic agent, a hormonal therapeutic agent, atoxin or an immunotherapeutic agent). The invention particularlyconcerns the embodiments of such pharmaceutical composition, whereinsuch one or more additional anti-cancer agents is a toxin selected fromthe group consisting of: a taxane, a maytansinoid, an auristatin, acalicheamicin, an anthracycline, a CC-1065 analog, docetaxel, acathepsin, ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin,RNase, and a toxic radioisotope.

The invention further pertains to a use of any of the above-describedisolated antibodies in the diagnosis of cancer, wherein the isolatedantibody is detectably labeled, and to any of the above-describedisolated detectably labeled antibodies for use in the diagnosis ofcancer. The invention particularly concerns the embodiments of such use,characterized in that the cancer is characterized by the presence of acancer cell selected from the group consisting of a cell of an adrenalgland tumor, an AIDS-associated cancer, an alveolar soft part sarcoma,an astrocytic tumor, bladder cancer, bone cancer, a brain and spinalcord cancer, a metastatic brain tumor, a breast cancer, a carotid bodytumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophoberenal cell carcinoma, a clear cell carcinoma, a colon cancer, acolorectal cancer, a cutaneous benign fibrous histiocytoma, adesmoplastic small round cell tumor, an ependymoma, a Ewing's tumor, anextraskeletal myxoid chondrosarcoma, a fibrogenesis imperfecta ossium, afibrous dysplasia of the bone, a gallbladder or bile duct cancer,gastric cancer, a gestational trophoblastic disease, a germ cell tumor,a head and neck cancer, hepatocellular carcinoma, an islet cell tumor, aKaposi's Sarcoma, a kidney cancer, a leukemia, a lipoma/benignlipomatous tumor, a liposarcoma/malignant lipomatous tumor, a livercancer, a lymphoma, a lung cancer, a medulloblastoma, a melanoma, ameningioma, a multiple endocrine neoplasia, a multiple myeloma, amyelodysplastic syndrome, a neuroblastoma, a neuroendocrine tumors, anovarian cancer, a pancreatic cancer, a papillary thyroid carcinoma, aparathyroid tumor, a pediatric cancer, a peripheral nerve sheath tumor,a phaeochromocytoma, a pituitary tumor, a prostate cancer, a posterioruveal melanoma, a rare hematologic disorder, a renal metastatic cancer,a rhabdoid tumor, a rhabdomysarcoma, a sarcoma, a skin cancer, asoft-tissue sarcoma, a squamous cell cancer, a stomach cancer, asynovial sarcoma, a testicular cancer, a thymic carcinoma, a thymoma, athyroid metastatic cancer, and a uterine cancer.

The invention further pertains to a use of any of the above-describedisolated antibodies or pharmaceutical compositions in the preparation ofa medicament for the treatment of cancer in a patient, and to amedicament for use in providing such treatment. The invention furtherpertains to such use, characterized in that the cancer is characterizedby the presence of a cancer cell selected from the group consisting of acell of an adrenal gland tumor, an AIDS-associated cancer, an alveolarsoft part sarcoma, an astrocytic tumor, bladder cancer, bone cancer, abrain and spinal cord cancer, a metastatic brain tumor, a breast cancer,a carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma,a chromophobe renal cell carcinoma, a clear cell carcinoma, a coloncancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, adesmoplastic small round cell tumor, an ependymoma, a Ewing's tumor, anextraskeletal myxoid chondrosarcoma, a fibrogenesis imperfecta ossium, afibrous dysplasia of the bone, a gallbladder or bile duct cancer,gastric cancer, a gestational trophoblastic disease, a germ cell tumor,a head and neck cancer, hepatocellular carcinoma, an islet cell tumor, aKaposi's Sarcoma, a kidney cancer, a leukemia, a lipoma/benignlipomatous tumor, a liposarcoma/malignant lipomatous tumor, a livercancer, a lymphoma, a lung cancer, a medulloblastoma, a melanoma, ameningioma, a multiple endocrine neoplasia, a multiple myeloma, amyelodysplastic syndrome, a neuroblastoma, a neuroendocrine tumors, anovarian cancer, a pancreatic cancer, a papillary thyroid carcinoma, aparathyroid tumor, a pediatric cancer, a peripheral nerve sheath tumor,a phaeochromocytoma, a pituitary tumor, a prostate cancer, a posterioruveal melanoma, a rare hematologic disorder, a renal metastatic cancer,a rhabdoid tumor, a rhabdomysarcoma, a sarcoma, a skin cancer, asoft-tissue sarcoma, a squamous cell cancer, a stomach cancer, asynovial sarcoma, a testicular cancer, a thymic carcinoma, a thymoma, athyroid metastatic cancer, and a uterine cancer.

The invention further pertains to any suchuses, characterized in thatthe use further comprises administration of one or more additionalcancer therapies selected from the group consisting of chemotherapy,immunotherapy, radiation therapy, hormonal therapy, and surgery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show the results of IHC investigations conducted usingnormal pancreas, liver, lung and colon tissue specimens with BRCA84D at0.625 μg/ml and 0.078 μg/ml (FIG. 1A) and normal heart, kidney andadrenal tissue with BRCA84D at 0.625 μg/ml (FIG. 1B).

FIG. 2 shows the results of IHC investigations conducted using cancerouspancreas, breast, colon and lung tissue specimens with BRCA84D at 0.625μg/ml and 0.078 μg/ml.

FIGS. 3A-3D show dose-dependent redirected killing mediated by theantibodies of the present invention. FIGS. 3A-3B show dose-dependentredirected killing of A498 renal carcinoma cells (with resting PBMC at18 hours (LDH)) by monoclonal antibodies reactive against B7-H3(effector:target ratio of 20:1) (FIG. 3A: BRCA68D, BRCA69D, PRCA157,GB8, TCR-4420; FIG. 3B: OVCA22, BRCA84D, TDH6, TES7, TCR-4420). FIGS.3C-3D show dose-dependent redirected killing of A549 lung cancer cells(with resting PBMC at 18 hours (LDH)) by monoclonal antibodies reactiveagainst B7-H3 (effector:target ratio of 30:1) (FIG. 3C: BRCA84D, OVCA22,PRCA157, TES7; FIG. 3D: TDH6, BRCA68D, BRCA69D).

FIGS. 4A-4B show the abilities of anti-B7-H3 antibodies to bind tosoluble B7H3-2Ig (FIG. 4A) and soluble B7H3-4Ig B7-H3 (FIG. 4B)(antibody concentration is 100 nM). Legend: (A) BLA8; (B) BRCA165; (C)BRCA68D; (D) BRCA69D; (E) BRCA84D; (F) GB8; (G) LUCA1; (H) LUCA50; (I)OVCA21; (J) OVCA22; (K) PA20; (L) PRCA123; (M) SG24; (N) SG27; (O) STO9;(P) TDH4 (184-192); (Q) TDH4; (R) TDH5; (S) TES7. The vertical positionof legend correlates with the position of the corresponding curve.

FIGS. 5A-5S demonstrate the binding affinity between antigens insolution and captured monoclonal antibodies (solid lines; B7-H3(4Ig) 100nM; dashed lines; B7-H3, 100 nM).

FIGS. 6A-6I show the results of BIACORE™ analyses of B7-H3 antibodiesimmobilized to B7-H3-2Ig (dashed gray lines) or B7-H3-4Ig (solid blacklines). Antibodies were titrated from 0.063 μM to 1 μM. Time is inseconds.

FIG. 7 provides a comparison BIACORE™ analysis of antibodies PRCA157,BRCA69D, BLA8, PA20, BRCA84D, GB8 and SG27.

FIG. 8 provides a BIACORE™ analysis demonstrating that antibodiesBRCA68D, BRCA69D, and PRCA157 do not compete with BRCA84D for binding tohuman B7-H3.

FIGS. 9A-9B show the results of studies on the ability of the anti-B7-H3antibodies of the present invention to become internalized upon bindingto cancer cells (FIG. 9A, prostate CSC cells; FIG. 9B, Hs700t pancreaticcells).

FIGS. 10A-10F show the ability of the anti-B7-H3 antibodies of thepresent invention to cross-block one another thereby revealingoverlapping or distinct epitopes. A ten-fold excess of competitorantibody was employed.

FIGS. 11A-11B show the alignment of the amino acid residues of thevariable light chains (FIG. 11A) or variable heavy chains (FIG. 11B) ofBRCA84D and its humanized derivative, hBRCA84D.

FIG. 12 shows the relative binding affinities of the hBRCA84D lightchain derivatives BRCA84D-3VL, BRCA84D-4VL and BRCA84D-5VL for humanB7-H3.

FIG. 13 shows the relative binding affinities of the hBRCA84D heavychain derivatives BRCA84D-2VH, BRCA84D-3VH and BRCA84D-4VH for humanB7-H3.

FIG. 14 shows the relative binding affinities of (1) antibodiescontaining hBRCA84D-2VL and hBRCA84D-2VH (trials 1 and 2), (2) chimericBRCA84D, (3) antibody containing hBRCA84D-5VL and chimeric BRCA84D-HCand (4) antibody containing hBRCA84D-5VL and hBRCA84D-2VH.

FIG. 15 shows the ability of Fc-modified humanized anti-B7-H3 antibodiesto inhibit tumor growth of HT-1197 urinary bladder carcinoma cells invivo in a murine xenograft model system. The Fc-modified hBRCA84D-2antibody (comprising Fc modifications L235V, F243L, R292P, Y300L, andP396L) was administered to the mice (at a dose of 1 μg/kg, 10 μg/kg, or20 μg/kg) 7 days, 14 days, 21 days and 28 days post implantation of thecancer cells.

FIG. 16 shows the ability of Fc-modified humanized anti-B7-H3 antibodiesto inhibit tumor growth of A498 renal carcinoma cells in vivo in amurine xenograft model system. The Fc-modified hBRCA84D-2 antibody(comprising Fc modifications L235V, F243L, R292P, Y300L, and P396L) wasadministered to the mice (at a dose of 1 μg/kg, 10 μg/kg, or 20 μg/kg) 7days, 14 days, 21 days and 28 days post implantation of the cancercells.

FIGS. 17A-17D demonstrate the ability of the hBRCA84D-2/anti-TCR DART(“T-DART”) to mediate redirected killing of SK-MES-1 lung cancer cells,A498 renal carcinoma cells, LNCaP prostate cancer cells, and UACC-62melanoma cells.

FIGS. 18A-18C show the pharmacokinetic decay of anti-B7-H3 Mab1 in thesera of male tumor-free male mCD16−/−, hCD16A_FOXN1 mice (FIGS.18A-18B). FIG. 18C shows the predicted pharmacokinetic profilesgenerated using a 2-compartment model with parameters from the 5 mg/kgdose at 0.1, 0.5, 1, 5, and 10 mg/kg.

FIG. 19 shows the relative expression of HER2 and PRCA135 by the HT-1197bladder cancer line.

FIG. 20 shows the binding affinity of anti-B7-H3 antibody hBRCA84Dvariants to HT-1197 cells.

FIGS. 21A-21C show the results of a murine xenograft analysis forHT-1197. Groups of 8 female mice received vehicle or 10 mg/kg IgGcontrol, or centuximab at a dose of 1, 5, or 15 mg/kg or anti-B7-H3antibody Mab1 at a dose of 0.1, 0.5, 1, 5, or 10 mg/kg (Q7D×5). Tumormeasurements were made every 3-4 days. FIG. 21A shows the ability ofanti-B7-H3 antibody Mab1 to prevent or inhibit tumor development in themurine xenograft model. Comparisons vs IgG control: Mab1 (1 and 5 mg/kg)vs IgG control *** from day 51; Mab1 (10 mg/kg) vs IgG control ** fromday 48. FIG. 21B shows the ability of centuximab to prevent or inhibittumor development in the murine xenograft model. Cetuximab (7 mg/kg) vsIgG control ** from day 51; Cetuximab (15 mg/kg) vs IgG control *** fromday 58. FIG. 21C compares the results obtained at the maximum dosestested.

FIGS. 22A-22B show the relative expression of HER2 and PMSA by theHT-1376 bladder cancer line.

FIG. 23 shows the results of a murine xenograft analysis for HT-1376.Groups of mice received vehicle or 1.0 mg/kg of anti-B7-H3 antibody Mab1(Q7D×4).

FIG. 24 show the results of a murine xenograft analysis for AGS. Groupsof mice received vehicle or 10 mg/kg anti-B7-H3 antibody Mab1 at a doseof 0.5, 1, 5, or 10 mg/kg (Q7D×5).

FIG. 25 show the results an in vitro cytotoxicity assay of A549 lungcancer cells upon incubation with hBRCA84D, chBRCA84D and hBRCA84 (FcVar1) variant anti-B7-H3 antibodies (E:T Ratio=25:1; Effector=HumanPBMC; LDH Assay readout).

FIG. 26 shows the results of a murine xenograft analysis for A549.Groups of mice received vehicle or 1.0 mg/kg of anti-B7-H3 antibody Mab1(Q7D×4).

FIG. 27 show the results of a murine xenograft analysis for CaLu3.Groups of mice received vehicle or 0.5, 1, or 5 mg/kg (Q7D×5) anti-B7-H3antibody Mab1 or IgG control (10 mg/ml).

FIGS. 28A-28C show the results of a murine xenograft analysis forLOX-IMVI melanoma cancer cells. Groups of 8 female mice received vehicleor 5 mg/kg IgG control, or Docetaxel at a dose of 5, 10 or 20 mg/kg oranti-B7-H3 antibody Mab1 at a dose of 0.5, 1, 5, or 10 mg/kg. FIG. 28Ashows the ability of anti-B7-H3 antibody Mab1 to prevent or inhibittumor development in the murine xenograft model. FIG. 28B shows theability of Docetaxel to prevent or inhibit tumor development in themurine xenograft model. FIG. 28C compares the results obtained at themaximum doses tested.

FIG. 29 shows the result of a murine xenograft analysis for UACC-62melanoma cancer cells. Groups of mice received vehicle or 5 mg/kg IgGcontrol, or anti-B7-H3 antibody Mab1 at a dose of 0.5, 1, 5, or 10mg/kg.

FIGS. 30A-30C show the results of a murine xenograft analysis for 2rvprostate cancer cells. Groups of 8 female mice received vehicle or 10mg/kg IgG control, or trastuzumab at a dose of 1, 7 or 15 mg/kg oranti-B7-H3 antibody Mab1 at a dose of 0.5, 1, 5, or 10 mg/kg. FIG. 30Ashows the ability of anti-B7-H3 antibody Mab1 to prevent or inhibittumor development in the murine xenograft model. FIG. 30B shows theability of trastuzumab to prevent or inhibit tumor development in themurine xenograft model. FIG. 30C compares the results obtained at themaximum doses tested.

FIG. 31 show the results an in vitro cytotoxicity assay of A498 renalcancer cells upon incubation with hBRCA84D, chBRCA84D and hBRCA84 (FcVar1) variant anti-B7-H3 antibodies (E:T Ratio=25:1; Effector=HumanPBMC; LDH Assay readout).

FIG. 32 shows the result of a murine xenograft analysis for A498 renalcancer cells. Groups of mice received vehicle or 10 mg/kg IgG control,or anti-B7-H3 antibody Mab1 at a dose of 0.1, 0.5, 1, 5, or 10 mg/kg.Centuximab (anti-EGRF antibody) was administered to a control group ofmice at doses of 1, 7, or 15 mg/kg.

FIGS. 33A-33B show the result of a murine xenograft analysis for 786-0renal cancer cells compared to centuximab. Groups of mice receivedvehicle or 10 mg/kg IgG control, or anti-B7-H3 antibody Mab1 at a doseof 0.1, 0.5, 1, 5, or 10 mg/kg. Centuximab (anti-EGRF antibody) wasadministered to a control group of mice at doses of 1, 7, or 15 mg/kg.

FIG. 34 shows the result of a murine xenograft analysis for 786-0 renalcancer cells compared to paclitaxel. Groups of mice received vehicle or5 mg/kg IgG control, or anti-B7-H3 antibody Mab1 at a dose of 0.1, 0.5,1, 5, or 10 mg/kg. Paclitaxel was administered to a control group ofeight such mice at a dose of 2.5 mg/kg.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to antibodies that are immunoreactive tothe mammalian, and more particularly, the human B7-H3 receptor and touses thereof, particularly in the treatment of cancer and inflammation.The invention thus particularly concerns humanized B7-H3-reactiveantibodies that are capable of mediating, and more preferably enhancingthe activation of the immune system against cancer cells that areassociated with a variety of human cancers.

I. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, MOLECULAR CLONING: ALABORATORY MANUAL, Third Edition (Sambrook et al. Eds., 2001) ColdSpring Harbor Press, Cold Spring Harbor, N.Y.; OLIGONUCLEOTIDESYNTHESIS: METHODS AND APPLICATIONS (Methods in Molecular Biology),Herdewijn, P., Ed., Humana Press, Totowa, N.J.; OLIGONUCLEOTIDESYNTHESIS (Gait, M. J., Ed., 1984); METHODS IN MOLECULAR BIOLOGY, HumanaPress, Totowa, N.J.; CELL BIOLOGY: A LABORATORY NOTEBOOK (Cellis, J. E.,Ed., 1998) Academic Press, New York, N.Y.; ANIMAL CELL CULTURE(Freshney, R. I., Ed., 1987); INTRODUCTION TO CELL AND TISSUE CULTURE(Mather, J. P. and Roberts, P. E., Eds., 1998) Plenum Press, New York,N.Y.; CELL AND TISSUE CULTURE: LABORATORY PROCEDURES (Doyle, A. et al.,Eds., 1993-8) John Wiley and Sons, Hoboken, N.J.; METHODS IN ENZYMOLOGY(Academic Press, Inc.) New York, N.Y.; WEIR'S HANDBOOK OF EXPERIMENTALIMMUNOLOGY (Herzenberg, L. A. et al. Eds. 1997) Wiley-BlackwellPublishers, New York, N.Y.; GENE TRANSFER VECTORS FOR MAMMALIAN CELLS(Miller, J. M. et al. Eds., 1987) Cold Spring Harbor Press, Cold SpringHarbor, N.Y.; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, F. M. etal., Eds., 1987) Greene Pub. Associates, New York, N.Y.; PCR: THEPOLYMERASE CHAIN REACTION, (Mullis, K. et al., Eds., 1994) Birkhäauser,Boston Mass.; CURRENT PROTOCOLS IN IMMUNOLOGY (Coligan, I. E. et al.,eds., 1991) John Wiley and Sons, Hoboken, N.J.; SHORT PROTOCOLS INMOLECULAR BIOLOGY (John Wiley and Sons, 1999) Hoboken, N.J.;IMMUNOBIOLOGY 7 (Janeway, C. A. et al. 2007) Garland Science, London,UK; Antibodies (P. Finch, 1997) Stride Publications, Devoran, UK;ANTIBODIES: A PRACTICAL APPROACH (D. Catty, ed., 1989) Oxford UniversityPress, USA, New York N.Y.); MONOCLONAL ANTIBODIES: A PRACTICAL APPROACH(Shepherd, P. et al. Eds., 2000) Oxford University Press, USA, New YorkN.Y.; USING ANTIBODIES: A LABORATORY MANUAL (Harlow, E. et al. Eds.,1998) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; THEANTIBODIES (Zanetti, M. et al. Eds. 1995) Harwood Academic Publishers,London, UK); and DEVITA, HELLMAN, AND ROSENBERG'S CANCER: PRINCIPLES &PRACTICE OF ONCOLOGY, EIGHTH EDITION, DeVita, V. et al. Eds. 2008,Lippincott Williams & Wilkins, Philadelphia, Pa.

II. Definitions

As used herein, the term “B7-H3” refers to a member of the human B7family of proteins, a type I membrane protein with Ig-like domains alsoknown as CD276. The term “2Ig-B7-H3” denotes the B7-H3 form thatcomprises only two Ig-like domains; the term “4Ig-B7-H3” denotes theB7-H3 form that comprises four Ig-like domains (see, Sun, M. et al.(2002) “Characterization of Mouse and Human B7-H3 Genes,” J. Immunol.168:6294-6297; Steinberger et al. (2004), “Molecular Characterization OfHuman 4Ig-B7-H3, A Member Of The B7 Family With Four Ig-Like Domains,”J. Immunol. 2004, 172(4):2352-2359 and Castriconi et al. (2004)“Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule ThatExerts A Protective Role From An NK Cell-Mediated Lysis,” Proc. Natl.Acad. Sci. (U.S.A.) 101(34):12640-12645). The antigen “TES7” (WO2008/066691) is an antigen sharing characteristics of the 4Ig-B7-H3.Accordingly, antibodies that specifically bind to TES7 bind to4Ig-B7-H3. The TES7 antigen may have more than one different epitope,and epitopes may be non-linear. Several anti-B7-H3 antibodies are knownto bind to non-linear epitopes, including some only present on the4Ig-B7-H3 isoform. It is currently believed that TEST may beover-expressed in certain cancer cells in comparison to their normaltissue counterparts.

Agonists, antagonists, and other modulators of B7-H3 function areexpressly included within the scope of this invention. These agonists,antagonists and modulators are polypeptides that comprise one or more ofthe antigenic determinant sites of B7-H3, or comprise one or morefragments of such sites, variants of such sites, or peptidomimetics ofsuch sites. These agonistic, antagonistic, and B7-H37 modulatorycompounds are provided in linear or cyclized form, and optionallycomprise at least one amino acid residue that is not commonly found innature or at least one amide isostere. These compounds may beglycosylated.

More specifically, the terms “B7-H3 modulator” as used herein aredefined as any compound that (1) is capable of disrupting or blockingthe interaction between human B7-H3 and its native ligands or ananti-B7-H3 antibody; (2) is capable of binding to human B7-H3 and itsnative ligands or an anti-B7-H3 antibody; (3) contains an antigenic sitethat can be used in the raising of antibodies capable of binding tohuman B7-H3 and its native ligands or an anti-B7-H3 antibody; (4)contains an antigenic site that can be used in the screening ofantibodies capable of binding to human B7-H3 and its native ligands oran anti-B7-H3 antibody; (5) contains an antigenic site that can be usedin the raising of antibodies capable of disrupting or blocking theinteraction between human B7-H3 and its native ligands or an anti-B7-H3antibody; (6) contains an antigenic site that can be used in thescreening of antibodies capable of disrupting or blocking theinteraction between human B7-H3 and its native ligands or an anti-B7-H3antibody. B7-H3 modulators may be “B7-H3 agonists” or “B7-H3antagonists” depending on whether their activity enhances T cellactivation or inhibits Tcell activation, respectively.

B7-H3 agonists, antagonists and modulators include B7-H3 variants, B7-H3peptide antagonists, peptidomimetics, and small molecules, anti-B7-H3antibodies and immunoglobulin variants, amino acid variants of humanB7-H3 including amino acid substitution, deletion, and additionvariants, or any combination thereof, and chimeric immunoglobulins. TheB7-H3 agonists, antagonists and modulators of this invention are basedon the identification of the B7-H3 domains involved in the binding ofhuman B7-H3 to its native ligands or anti-B7-H3 antibodies. Thus, theinvention provides B7-H3 agonists, antagonists and modulators withmolecular structures that duplicate or mimic one or more of theanti-B7-H3 binding domains of human B7-H3.

As used herein, the term “B7-H3 variant” denotes any amino acid variantof human B7-H3, including amino acid substitution, deletion, andaddition variants, or any combination thereof. The definitionencompasses chimeric molecules such as human B7-H3/non-human chimerasand other hybrid molecules. Also included in the definition is anyfragment of a B7-H3 variant molecule that comprises the variant orhybrid region(s) of the molecule.

As used herein, an “antibody” is an immunoglobulin molecule capable ofspecific binding to a target, such as a carbohydrate, polynucleotide,lipid, polypeptide, etc., through at least one antigen recognition site,located in the variable region of the immunoglobulin molecule. As usedherein, the term encompasses polyclonal or monoclonal antibodies, fusionproteins comprising an antibody portion with an antigen recognition siteof the required specificity, humanized antibodies, chimeric antibodies,“BiTEs,” “DART” molecules and any other modified configuration of theimmunoglobulin molecule that comprises an antibody chain of the requiredspecificity.

The term “BiTEs” (bi-specific T-cell engagers) refers to a singlepolypeptide chain molecule that having two antigen binding domains, oneof which binds to a T-cell antigen and the second of which binds to anantigen present on the surface of a target (WO 05/061547; Baeuerle, P etal. (2008) “BITE®: A New Class Of Antibodies That Recruit T Cells,”Drugs of the Future 33: 137-147; Bargou, et al. 2008) “Tumor Regressionin Cancer Patients by Very Low Doses of a T Cell-Engaging Antibody,”Science 321: 974-977).

The term “DART™” (dual affinity retargeting reagent) refers to animmunoglobulin molecule that comprises at least two polypeptide chainsthat associate (especially through a covalent interaction) to form atleast two epitope binding sites, which may recognize the same ordifferent epitopes. Each of the polypeptide chains of a DART™ comprisean immunoglobulin light chain variable region and an immunoglobulinheavy chain variable region, but these regions do not interact to forman epitope binding site. Rather, the immunoglobulin heavy chain variableregion of one (e.g., the first) of the DART™ polypeptide chainsinteracts with the immunoglobulin light chain variable region of adifferent (e.g., the second) DART™ polypeptide chain to form an epitopebinding site. Similarly, the immunoglobulin light chain variable regionof one (e.g., the first) of the DART™ polypeptide chains interacts withthe immunoglobulin heavy chain variable region of a different (e.g., thesecond) DART™ polypeptide chain to form an epitope binding site. DART™smay be monospecific, bispecific, trispecific, etc., thus being able tosimultaneously bind one, two, three or more different epitopes (whichmay be of the same or of different antigens). DART™s may additionally bemonovalent, bivalent, trivalent, tetravalent, pentavalent, hexavelent,etc., thus being able to simultaneously bind one, two, three, four,five, six or more molecules. These two attributes of DART™s (i.e.,degree of specificity and valency may be combined, for example toproduce bispecific antibodies (i.e., capable of binding two epitopes)that are tetravalent (i.e., capable of binding four sets of epitopes),etc. DART™ molecules are disclosed in PCT Publications WO 2006/113665,WO 2008/157379, and WO 2010/080538.

The term “monoclonal antibody” refers to a homogeneous antibodypopulation wherein the monoclonal antibody is comprised of amino acids(naturally occurring and non-naturally occurring) that are involved inthe selective binding of an antigen. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site. The term“monoclonal antibody” encompasses not only intact monoclonal antibodiesand full-length monoclonal antibodies, but also fusion proteinscomprising an antibody portion, humanized monoclonal antibodies,chimeric monoclonal antibodies, and any other modified configuration ofthe immunoglobulin molecule that comprises an antigen recognition siteof the required specificity and the ability to bind to an antigen. It isnot intended to be limited as regards to the source of the antibody orthe manner in which it is made (e.g., by hybridoma, phage selection,recombinant expression, transgenic animals, etc.).

The term “humanized antibody” refer to a chimeric molecule, generallyprepared using recombinant techniques, having an antigen binding sitederived from an immunoglobulin from a non-human species and theremaining immunoglobulin structure of the molecule based upon thestructure and/or sequence of a human immunoglobulin. The antigen-bindingsite may comprise either complete variable domains fused onto constantdomains or only the complementarity determining regions (CDRs) graftedonto appropriate framework regions in the variable domains. Antigenbinding sites may be wild type or modified by one or more amino acidsubstitutions. This eliminates the constant region as an immunogen inhuman individuals, but the possibility of an immune response to theforeign variable region remains (LoBuglio, A. F. et al. (1989)“Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And ImmuneResponse,” Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224). Anotherapproach focuses not only on providing human-derived constant regions,but modifying the variable regions as well so as to reshape them asclosely as possible to human form. It is known that the variable regionsof both heavy and light chains contain three complementarity-determiningregions (CDRs) which vary in response to the antigens in question anddetermine binding capability, flanked by four framework regions (FRs)which are relatively conserved in a given species and which putativelyprovide a scaffolding for the CDRs. When nonhuman antibodies areprepared with respect to a particular antigen, the variable regions canbe “reshaped” or “humanized” by grafting CDRs derived from nonhumanantibody on the FRs present in the human antibody to be modified.Application of this approach to various antibodies has been reported bySato, K. et al. (1993) Cancer Res 53:851-856. Riechmann, L. et al.(1988) “Reshaping Human Antibodies for Therapy,” Nature 332:323-327;Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies: Grafting AnAntilysozyme Activity,” Science 239:1534-1536; Kettleborough, C. A. etal. (1991) “Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting:The Importance Of Framework Residues On Loop Conformation,” ProteinEngineering 4:773-3783; Maeda, H. et al. (1991) “Construction OfReshaped Human Antibodies With HIV-Neutralizing Activity,” HumanAntibodies Hybridoma 2:124-134; Gorman, S. D. et al. (1991) “Reshaping ATherapeutic CD4 Antibody,” Proc. Natl. Acad. Sci. (U.S.A.) 88:4181-4185;Tempest, P. R. et al. (1991) “Reshaping A Human Monoclonal Antibody ToInhibit Human Respiratory Syncytial Virus Infection in vivo,”Bio/Technology 9:266-271; Co, M. S. et al. (1991) “Humanized AntibodiesFor Antiviral Therapy,” Proc. Natl. Acad. Sci. (U.S.A.) 88:2869-2873;Carter, P. et al. (1992) “Humanization Of An Anti-p185her2 Antibody ForHuman Cancer Therapy,” Proc. Natl. Acad. Sci. (U.S.A.) 89:4285-4289; andCo, M. S. et al. (1992) “Chimeric And Humanized Antibodies WithSpecificity For The CD33 Antigen,” J. Immunol. 148:1149-1154. In someembodiments, humanized antibodies preserve all CDR sequences (forexample, a humanized mouse antibody which contains all six CDRs from themouse antibodies). In other embodiments, humanized antibodies have oneor more CDRs (one, two, three, four, five, six) which are altered withrespect to the original antibody, which are also termed one or more CDRs“derived from” one or more CDRs from the original antibody.

As used herein, an antibody or a polypeptide is said to “specifically”bind a region of another molecule (i.e., an epitope) if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with that epitope relative to alternativeepitopes. For example, an antibody that specifically binds to a B7-H3epitope is an antibody that binds this B7-H3 epitope with greateraffinity, avidity, more readily, and/or with greater duration than itbinds to other B7-H3 epitopes or non-B7-H3 epitopes. It is alsounderstood by reading this definition that, for example, an antibody (ormoiety or epitope) that specifically binds to a first target may or maynot specifically or preferentially bind to a second target. As such,“specific binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means “specific” binding.

As used herein, the term “immunologically active” in reference to anepitope being or “remaining immunologically active” refers to theability of an antibody (e.g., an anti-B7-H3 antibody) to bind to theepitope under different conditions, for example, after the epitope hasbeen subjected to reducing and denaturing conditions.

Different biological functions are associated with anti-B7-H3antibodies, including, but not limited to one or more of: an ability tospecifically bind to B7-H3 (and in particular B7-H3 molecules that areexpressed on the surfaces of cancer cells, including but not limited tokidney, prostate, or lung, cancer cells); an ability to competitivelyinhibits preferential binding of a known anti-B7-H3 antibody to B7-H3,including the ability to preferentially bind to the same B7-H3 epitopeto which the original antibody preferentially binds; an ability to bindto a portion of B7-H3 that is exposed on the surface of a living cell invitro or in vivo; an ability to bind to a portion of B7-H3 that isexposed on the surface of living cancer cells, such as but not limitedto prostate, lung or kidney cancer cells; an ability to deliver achemotherapeutic agent to cancerous cells (such as kidney, prostate, orlung cancer cells) expressing B7-H3 on their surface; and/or an abilityto deliver a therapeutic agent or detectable marker into cancer cellsexpressing B7-H3 on their surface. As discussed herein, polypeptides(including antibodies) of the invention may have any one or more ofthese characteristics.

An “anti-B7-H3 equivalent antibody” or “anti-B7-H3 equivalentpolypeptide” refers to an antibody or a polypeptide having one or morebiological functions associated with an anti-B7-H3 antibody, such as,for example binding specificity.

As used herein, the term “agent” refers to a biological, pharmaceutical,or chemical compound. Non-limiting examples include simple or complexorganic or inorganic molecule, a peptide, a protein, an oligonucleotide,an antibody, an antibody derivative, antibody fragment, a vitaminderivative, a carbohydrate, a toxin, or a chemotherapeutic compound.Various compounds can be synthesized, for example, small molecules andoligomers (e.g., oligopeptides and oligonucleotides), and syntheticorganic compounds based on various core structures. In addition, variousnatural sources can provide compounds for screening, such as plant oranimal extracts, and the like.

Agents that are employed in the methods of this invention can berandomly selected or rationally selected or designed. As used herein, anagent is said to be randomly selected when the agent is chosen withoutprior consideration or knowledge of the specific amino acid or otherchemical moieties involved in the association of the molecule with itsnative binding partner(s) or known antibodies. An example of a randomlyselected agent is an agent that is identified through the use andscreening of a chemical library or a peptide combinatorial library.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a non-random basis that takes into accountthe sequence of the target site and/or its conformation in connectionwith the agent's action. With respect to anti-B7-H3 agents, it iscurrently believed that there are at least three epitopes on B7-H3against which antibodies can be raised and therefore at least threesites of action for agents that block B7-H3/anti-B7-H3 interaction. Thisinvention also encompasses agents that act at the sites of interactionbetween B7-H3 and its native binding partner, although other ligands andtheir active B7-H3-interactive sites are also encompassed within thescope of this invention, whether currently known or later identified.Agents can be rationally selected or rationally designed by utilizingthe peptide sequences that make up the contact sites of thereceptor/ligand and/or B7-H3/anti-B7-H3 antibody complex. For example, arationally selected peptide agent can be a peptide whose amino acidsequence is identical to an epitope appearing on B7-H3 as it is exposedon the surface of a living cell in its native environment. Such an agentwill reduce or block the association of the anti-B7-H3 antibody withB7-H3, or the association of B7-H3 with its native ligand, as desired,by binding to the anti-B7-H3 antibody or to the native ligand.

As used herein, the term “labeled,” with regard to an antibody, isintended to encompass direct labeling of the antibody by coupling (i.e.,physically linking) a detectable substance, such as a radioactive agentor a fluorophore (e.g. phycoerythrin (PE) or fluorescein isothiocyanate(also known as fluoroisothiocyanate or FITC)) to the antibody, as wellas indirect labeling of the probe or antibody by reactivity with adetectable substance.

As used herein, the term “association”, with regard to an antibody,includes covalent and non-covalent attachment or binding of an agent(e.g., chemotherapeutic agent) to the antibody. The antibody can beassociated with an agent (e.g., chemotherapeutic agent) by directbinding or indirect binding via attachment to a common platform, suchthat the antibody directs the localization of the agent to the cancerouscell to which the antibody binds and wherein the antibody and agent donot substantially dissociate under physiological conditions such thatthe agent is not targeted to the same cancerous cell to which theantibody binds or such that the agent's potency is not decreased.

The term “biological sample” encompasses a variety of sample typesobtained from an individual and can be used in a diagnostic ormonitoring assay. The definition encompasses saliva, blood and otherliquid samples of biological origin, solid tissue samples such as abiopsy specimen or tissue cultures or cells derived therefrom, and theprogeny thereof, for example, cells obtained from a tissue samplecollected from an individual suspected of having cancer, in preferredembodiments from ovary, lung, prostate, pancreas, colon, and breasttissue. The definition also includes samples that have been manipulatedin any way after their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides, or embedding in a semi-solid or solid matrix forsectioning purposes. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

The term “host cell” includes an individual cell or cell culture thatcan be or has been a recipient for vector(s) for incorporation ofpolynucleotide inserts. Host cells include progeny of a single hostcell, and the progeny may not necessarily be completely identical (inmorphology or in genomic DNA complement) to the original parent cell dueto natural, accidental, or deliberate mutation. A host cell includescells transfected in vivo with a polynucleotide(s) of this invention.

As used herein, the term “delaying development of metastasis” means todefer, hinder, slow, retard, stabilize, and/or postpone development ofmetastasis. This delay can be of varying lengths of time, depending onthe history of the cancer and/or individual being treated. As is evidentto one skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developthe metastasis.

As used herein, an “effective amount” of a pharmaceutical composition,in one embodiment, is an amount sufficient to effect beneficial ordesired results including, without limitation, clinical results such asshrinking the size of the tumor (in the cancer context, for example,breast or prostate cancer), retardation of cancerous cell growth,delaying the development of metastasis, decreasing symptoms resultingfrom the disease, increasing the quality of life of those suffering fromthe disease, decreasing the dose of other medications required to treatthe disease, enhancing the effect of another medication such as viatargeting and/or internalization, delaying the progression of thedisease, and/or prolonging survival of individuals. An effective amountcan be administered in one or more administrations. For purposes of thisinvention, an effective amount of drug, compound, or pharmaceuticalcomposition is an amount sufficient to reduce the proliferation of (ordestroy) cancerous cells and to reduce and/or delay the development, orgrowth, of metastases of cancerous cells, either directly or indirectly.In some embodiments, an effective amount of a drug, compound, orpharmaceutical composition may or may not be achieved in conjunctionwith another drug, compound, or pharmaceutical composition. Thus, an“effective amount” may be considered in the context of administering oneor more chemotherapeutic agents, and a single agent may be considered tobe given in an effective amount if, in conjunction with one or moreother agents, a desirable result may be or is achieved. While individualneeds vary, determination of optimal ranges of effective amounts of eachcomponent is within the skill of the art. Typical dosages comprise 0.1-to 100 mg/kg/body weight. The preferred dosages comprise 1 to 100mg/kg/body weight. The most preferred dosages comprise 10 to 100mg/kg/body weight.

As used herein, a nucleic acid molecule or agent, antibody, compositionor cell, etc., is said to be “isolated” when that nucleic acid molecule,agent, antibody, composition, or cell, etc. is substantially separatedfrom contaminant nucleic acid molecules, antibodies, agents,compositions, or cells, etc. naturally present in its original source.

The term “individual” refers to a vertebrate animal, preferably a mammalMammals include, but are not limited to, humans, farm animals, sportanimals, pets, primates, mice and rats. In the most preferredembodiment, the term individual denotes a human.

The terms “polypeptide,” “oligopeptide,” “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon an antibody, the polypeptides can occur as single chains or asassociated chains.

Also encompassed within the scope of the invention are peptidomimeticsof the B7-H3 peptide agonists, antagonists and modulators (includinganti-B7-H3 antibodies) described herein. Such peptidomimetics includepeptides wherein at least one amino acid residue is substituted with anamino acid residue that is not commonly found in nature, such as the Disomer of the amino acid or an N-alkylated species of the amino acid. Inother embodiments, peptidomimetics are constructed by replacing at leastone amide bond (—C(═O)—NH—) in a B7-H3 peptide agonist, antagonist ormodulators with an amide isostere. Suitable amide isosteres include—CH₂—NH—, —CH₂—S—, —CH₂—S(O)—, —CH₂—S(O)₂—, —CH₂—CH₂—, —CH═CH— (E or Zform), —C(═O)—CH₂—, —CH(CN)—NH—, —C(OH)—CH₂—, and —O—C(═O)—NH—. Theamide bonds in a B7-H3 peptide agonist, antagonist or modulator that aresuitable candidates for replacement with amide isosteres include bondsthat are hydrolyzable by the endogenous esterases or proteases of theintended subject of B7-H3 peptide agonist, antagonist or modulatortreatment.

As used herein, the term “substantially pure” refers to material that isat least 50% pure (i.e., free from contaminants), more preferably atleast 90% pure, more preferably at least 95% pure, more preferably atleast 98% pure, more preferably at least 99% pure, and most preferablygreater than 99% pure.

As used herein, the term “toxin” refers to any substance, which effectsan adverse response within a cell. For example, a toxin directed to acancerous cell would have an adverse, sometimes deleterious effect, onthe cancerous cell. Examples of toxins include, but are not limited to,a taxane, a maytansinoid, an auristatin (e.g., monomethyl auristatin(MMAE), monomethyl auristatin F (MMAF), auristatin E (AE), etc.) (suchas those disclosed in U.S. Pat. Nos. 5,208,020; 5,416,064; 6,333,410;6,340,701; 6,372,738; 6,436,931; 6,441,163; 6,596,757; 7,276,497;7,585,857; or 7,851,432), a calicheamicin, an anthracycline (e.g.,doxorubicin), a CC-1065 analog, docetaxel; cathepsin B or E; ricin,gelonin, Pseudomonas exotoxin, diphtheria toxin, and RNase; radiolabeledantibodies (e.g., tiuxetan-conjugated or labeled with a toxicradioisotope (for example, ⁹⁰Y; ¹³¹I, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, ²¹³Bi,²²⁵Ac, etc.).

As used herein, the terms “treatment” or “treating” denote an approachfor obtaining a beneficial or desired result including and preferably abeneficial or desired clinical result. Such beneficial or desiredclinical results include, but are not limited to, one or more of thefollowing: reducing the proliferation of (or destroying) cancerous cellsor other diseased, reducing metastasis of cancerous cells found incancers, shrinking the size of the tumor, decreasing symptoms resultingfrom the disease, increasing the quality of life of those suffering fromthe disease, decreasing the dose of other medications required to treatthe disease, delaying the progression of the disease, and/or prolongingsurvival of individuals.

As used herein, the term cancer is intended to encompass cancerscharacterized by the presence of a cancer cell selected from the groupconsisting of a cell of an adrenal gland tumor, an AIDS-associatedcancer, an alveolar soft part sarcoma, an astrocytic tumor, bladdercancer (squamous cell carcinoma and transitional cell carcinoma), bonecancer (adamantinoma, aneurismal bone cysts, osteochondroma,osteosarcoma), a brain and spinal cord cancer, a metastatic brain tumor,a breast cancer, a carotid body tumors, a cervical cancer, achondrosarcoma, a chordoma, a chromophobe renal cell carcinoma, a clearcell carcinoma, a colon cancer, a colorectal cancer, a cutaneous benignfibrous histiocytoma, a desmoplastic small round cell tumor, anependymoma, a Ewing's tumor, an extraskeletal myxoid chondrosarcoma, afibrogenesis imperfecta ossium, a fibrous dysplasia of the bone, agallbladder or bile duct cancer, gastric cancer, a gestationaltrophoblastic disease, a germ cell tumor, a head and neck cancer,hepatocellular canrcinoma, an islet cell tumor, a Kaposi's Sarcoma, akidney cancer (nephroblastoma, papillary renal cell carcinoma), aleukemia, a lipoma/benign lipomatous tumor, a liposarcoma/malignantlipomatous tumor, a liver cancer (hepatoblastoma, hepatocellularcarcinoma), a lymphoma, a lung cancer, a medulloblastoma, a melanoma, ameningioma, a multiple endocrine neoplasia, a multiple myeloma, amyelodysplastic syndrome, a neuroblastoma, a neuroendocrine tumors, anovarian cancer, a pancreatic cancer, a papillary thyroid carcinoma, aparathyroid tumor, a pediatric cancer, a peripheral nerve sheath tumor,a phaeochromocytoma, a pituitary tumor, a prostate cancer, a posterioruveal melanoma, a rare hematologic disorder, a renal metastatic cancer,a rhabdoid tumor, a rhabdomysarcoma, a sarcoma, a skin cancer, asoft-tissue sarcoma, a squamous cell cancer, a stomach cancer, asynovial sarcoma, a testicular cancer, a thymic carcinoma, a thymoma, athyroid metastatic cancer, and a uterine cancer (carcinoma of thecervix, endometrial carcinoma, and leiomyoma).

III. Methods of Making Antibodies and Polypeptides

Methods of making monoclonal antibodies are known in the art. One methodwhich may be employed is the method of Kohler, G. et al. (1975)“Continuous Cultures Of Fused Cells Secreting Antibody Of PredefinedSpecificity,” Nature 256:495-497 or a modification thereof. Typically,monoclonal antibodies are developed in non-human species, such as mice.In general, a mouse or rat is used for immunization but other animalsmay also be used. The antibodies are produced by immunizing mice with animmunogenic amount of cells, cell extracts, or protein preparations thatcontain human B7-H3. The immunogen can be, but is not limited to,primary cells, cultured cell lines, cancerous cells, nucleic acids, ortissue. In one embodiment, human lung carcinoma cells are used. Cellsused for immunization, for example, human testis or pancreaticadenocarcinoma or stomach cells, may be cultured for a period of time(e.g., at least 24 hours) prior to their use as an immunogen. Cells(e.g., human testis, stomach, or pancreatic adenocarcinoma cells) may beused as immunogens by themselves or in combination with a non-denaturingadjuvant, such as Ribi. In general, cells should be kept intact andpreferably viable when used as immunogens. Intact cells may allowantigens to be better detected than ruptured cells by the immunizedanimal. Use of denaturing or harsh adjuvants, e.g., Freud's adjuvant,may rupture cells and therefore is discouraged. The immunogen may beadministered multiple times at periodic intervals such as, bi weekly, orweekly, or may be administered in such a way as to maintain viability inthe animal (e.g., in a tissue recombinant).

In one embodiment, monoclonal antibodies that bind to B7-H3 are obtainedby using host cells that over-express B7-H3 as an immunogen. Such cellsinclude, by way of example and not by limitation, human lung carcinomacells and human colon cancer cells.

To monitor the antibody response, a small biological sample (e.g.,blood) may be obtained from the animal and tested for antibody titeragainst the immunogen. The spleen and/or several large lymph nodes canbe removed and dissociated into single cells. If desired, the spleencells may be screened (after removal of non-specifically adherent cells)by applying a cell suspension to a plate or to a well coated with theantigen. B-cells, expressing membrane-bound immunoglobulin specific forthe antigen, will bind to the plate, and are not rinsed away with therest of the suspension. Resulting B-cells, or all dissociated spleencells, can then be fused with myeloma cells (e.g., X63-Ag8.653 and thosefrom the SaIk Institute, Cell Distribution Center, San Diego, Calif.).Polyethylene glycol (PEG) may be used to fuse spleen or lymphocytes withmyeloma cells to form a hybridoma. The hybridoma is then cultured in aselective medium (e.g., hypoxanthine, aminopterin, thymidine medium,otherwise known as “HAT medium”). The resulting hybridomas are thenplated by limiting dilution, and are assayed for the production ofantibodies that bind specifically to the immunogen, using, for example,FACS (fluorescence activated cell sorting) or immunohistochemistry (IHC)screening. The selected monoclonal antibody-secreting hybridomas arethen cultured either in vitro (e.g., in tissue culture bottles or hollowfiber reactors), or in vivo (e.g., as ascites in mice).

As another alternative to the cell fusion technique, Epstein-Barr Virus(EBV)-immortalized B cells may be used to produce monoclonal antibodiesof the subject invention. The hybridomas are expanded and subcloned, ifdesired, and supernatants are assayed for anti-immunogen activity byconventional assay procedures (e.g., FACS, IHC, radioimmunoassay, enzymeimmunoassay, fluorescence immunoassay, etc.).

In another alternative, anti-B7-H3 monoclonal antibody and any otherequivalent antibodies can be sequenced and produced recombinantly by anymeans known in the art (e.g., humanization, use of transgenic mice toproduce fully human antibodies, phage display technology, etc.). In oneembodiment, anti-B7-H3 monoclonal antibody is sequenced and thepolynucleotide sequence is then cloned into a vector for expression orpropagation. The sequence encoding the antibody of interest may bemaintained in a vector in a host cell and the host cell can then beexpanded and frozen for future use.

The polynucleotide sequence of anti-B7-H3 monoclonal antibody and anyother equivalent antibodies may be used for genetic manipulation togenerate a “humanized” antibody, to improve the affinity, or othercharacteristics of the antibody. The general principle in humanizing anantibody involves retaining the basic sequence of the antigen-bindingportion of the antibody, while swapping the non-human remainder of theantibody with human antibody sequences. There are four general steps tohumanize a monoclonal antibody. These are: (1) determining thenucleotide and predicted amino acid sequence of the starting antibodylight and heavy variable domains (2) designing the humanized antibody,i.e., deciding which antibody framework region to use during thehumanizing process (3) the actual humanizing methodologies/techniquesand (4) the transfection and expression of the humanized antibody. See,for example, U.S. Pat. Nos. 4,816,567; 5,807,715; 5,866,692; and6,331,415.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent or modified rodent V regionsand their associated complementarity determining regions (CDRs) fused tohuman constant domains (see, for example, Winter et al. (1991) “Man-madeAnditbodies,” Nature 349:293-299; Lobuglio et al. (1989) “Mouse/HumanChimeric Monoclonal Antibody In Man: Kinetics And Immune Response,”Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224 (1989), Shaw et al. (1987)“Characterization Of A Mouse/Human Chimeric Monoclonal Antibody (17-1A)To A Colon Cancer Tumor-Associated Antigen,” J. Immunol. 138:4534-4538,and Brown et al. (1987) “Tumor-Specific Genetically EngineeredMurine/Human Chimeric Monoclonal Antibody,” Cancer Res. 47:3577-3583).Other references describe rodent CDRs grafted into a human supportingframework region (FR) prior to fusion with an appropriate human antibodyconstant domain (see, for example, Riechmann, L et al. (1988) “ReshapingHuman Antibodies for Therapy,” Nature 332:323-327; Verhoeyen, M. et al.(1988) “Reshaping Human Antibodies: Grafting An Antilysozyme Activity,”Science 239:1534-1536; and Jones et al. (1986) “Replacing TheComplementarity-Determining Regions In A Human Antibody With Those FromA Mouse,” Nature 321:522-525). Another reference describes rodent CDRssupported by recombinantly veneered rodent framework regions. See, forexample, European Patent Publication No. 519,596. These “humanized”molecules are designed to minimize unwanted immunological responsetoward rodent anti-human antibody molecules, which limits the durationand effectiveness of therapeutic applications of those moieties in humanrecipients. Other methods of humanizing antibodies that may also beutilized are disclosed by Daugherty et al. (1991) “Polymerase ChainReaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression OfA Murine Monoclonal Antibody Directed Against The CD18 Component OfLeukocyte Integrins,” Nucl. Acids Res. 19:2471-2476 and in U.S. Pat.Nos. 6,180,377; 6,054,297; 5,997,867; and 5,866,692.

The invention also encompasses single chain variable region fragments(“scFv”) of antibodies of this invention, such as mu-anti-B7-H3. Singlechain variable region fragments are made by linking light and/or heavychain variable regions by using a short linking peptide. Bird et al.(1988) (“Single-Chain Antigen-Binding Proteins,” Science 242:423-426)describes example of linking peptides which bridge approximately 3.5 nmbetween the carboxy terminus of one variable region and the aminoterminus of the other variable region. Linkers of other sequences havebeen designed and used (Bird et al. (1988) “Single-Chain Antigen-BindingProteins,” Science 242:423-426). Linkers can in turn be modified foradditional functions, such as attachment of drugs or attachment to solidsupports. The single chain variants can be produced either recombinantlyor synthetically. For synthetic production of scFv, an automatedsynthesizer can be used. For recombinant production of scFv, a suitableplasmid containing polynucleotide that encodes the scFv can beintroduced into a suitable host cell, either eukaryotic, such as yeast,plant, insect or mammalian cells, or prokaryotic, such as E. coli.Polynucleotides encoding the scFv of interest can be made by routinemanipulations such as ligation of polynucleotides. The resultant scFvcan be isolated using standard protein purification techniques known inthe art.

The invention includes modifications to antibodies and polypeptides thatbind to B7-H3 and its agonists, antagonists, and modulators, includingfunctionally equivalent antibodies and polypeptides that do notsignificantly affect their properties and variants that have enhanced ordecreased activity. Modification of polypeptides is routine practice inthe art and need not be described in detail herein. Examples of modifiedpolypeptides include polypeptides with conservative substitutions ofamino acid residues, one or more deletions or additions of amino acidswhich do not significantly deleteriously change the functional activity,or use of chemical analogs Amino acid residues which can beconservatively substituted for one another include but are not limitedto: glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine;aspartic acid/glutamic acid; serine/threonine; lysine/arginine; andphenylalanine/tryosine. These polypeptides also include glycosylated andnonglycosylated polypeptides, as well as polypeptides with otherpost-translational modifications, such as, for example, glycosylationwith different sugars, acetylation, and phosphorylation. Preferably, theamino acid substitutions would be conservative, i.e., the substitutedamino acid would possess similar chemical properties as that of theoriginal amino acid. Such conservative substitutions are known in theart, and examples have been provided above Amino acid modifications canrange from changing or modifying one or more amino acids to completeredesign of a region, such as the variable region. Changes in thevariable region can alter binding affinity and/or specificity. Othermethods of modification include using coupling techniques known in theart, including, but not limited to, enzymatic means, oxidativesubstitution and chelation. Modifications can be used, for example, forattachment of labels for immunoassay, such as the attachment ofradioactive moieties for radioimmunoassay. Modified polypeptides aremade using established procedures in the art and can be screened usingstandard assays known in the art.

The invention also encompasses fusion proteins comprising one or more ofthe polypeptides or antibodies of this invention. In one embodiment, afusion polypeptide is provided that comprises a light chain, a heavychain or both a light and heavy chain. In another embodiment, the fusionpolypeptide contains a heterologous immunoglobulin constant region. Inanother embodiment, the fusion polypeptide contains a light chainvariable region and a heavy chain variable region of an antibodyproduced from a publicly-deposited hybridoma. For purposes of thisinvention, an antibody fusion protein contains one or more polypeptidedomains that specifically bind to B7-H3 and another amino acid sequenceto which it is not attached in the native molecule, for example, aheterologous sequence or a homologous sequence from another region.

An anti-B7-H3 polypeptide, and other B7-H3 agonists, antagonists andmodulators can be created by methods known in the art, for example,synthetically or recombinantly One method of producing B7-H3 peptideagonists, antagonists and modulators involves chemical synthesis of thepolypeptide, followed by treatment under oxidizing conditionsappropriate to obtain the native conformation, that is, the correctdisulfide bond linkages. This can be accomplished using methodologieswell known to those skilled in the art (see, e.g., Kelley, R. F. et al.(1990) In: GENETIC ENGINEERING PRINCIPLES AND METHODS, Setlow, J. K.Ed., Plenum Press, N.Y., vol. 12, pp 1-19; Stewart, J. M et al. (1984)SOLID PHASE PEPTIDE SYNTHESIS, Pierce Chemical Co., Rockford, Ill.; seealso U.S. Pat. Nos. 4,105,603; 3,972,859; 3,842,067; and 3,862,925).

Polypeptides of the invention may be conveniently prepared using solidphase peptide synthesis (Merrifield, B. (1986) “Solid Phase Synthesis,”Science 232(4748):341-347; Houghten, R. A. (1985) “General Method ForThe Rapid Solid-Phase Synthesis Of Large Numbers Of Peptides:Specificity Of Antigen Antibody Interaction At The Level Of IndividualAmino Acids,” Proc. Natl. Acad. Sci. (U.S.A.) 82(15):5131-5135; Ganesan,A. (2006) “Solid-Phase Synthesis In The Twenty-First Century,” Mini Rev.Med. Chem. 6(1):3-10).

In yet another alternative, fully human antibodies may be obtainedthrough the use of commercially available mice that have been engineeredto express specific human immunoglobulin proteins. Transgenic animalsthat are designed to produce a more desirable (e.g., fully humanantibodies) or more robust immune response may also be used forgeneration of humanized or human antibodies. Examples of such technologyare XENOMOUSE™ (Abgenix, Inc., Fremont, Calif.) and HUMAB-MOUSE® and TCMOUSE™ (both from Medarex, Inc., Princeton, N.J.).

In an alternative, antibodies may be made recombinantly and expressedusing any method known in the art. Antibodies may be made recombinantlyby first isolating the antibodies made from host animals, obtaining thegene sequence, and using the gene sequence to express the antibodyrecombinantly in host cells (e.g., CHO cells). Another method that maybe employed is to express the antibody sequence in plants {e.g.,tobacco) or transgenic milk. Suitable methods for expressing antibodiesrecombinantly in plants or milk have been disclosed (see, for example,Peeters et al. (2001) “Production Of Antibodies And Antibody FragmentsIn Plants,” Vaccine 19:2756; Lonberg, N. et al. (1995) “Human AntibodiesFrom Transgenic Mice,” Int. Rev. Immunol 13:65-93; and Pollock et al.(1999) “Transgenic Milk As A Method For The Production Of RecombinantAntibodies,” J. Immunol Methods 231:147-157). Suitable methods formaking derivatives of antibodies, e.g., humanized, single chain, etc.are known in the art. In another alternative, antibodies may be maderecombinantly by phage display technology (see, for example, U.S. Pat.Nos. 5,565,332; 5,580,717; 5,733,743; 6,265,150; and Winter, G. et al.(1994) “Making Antibodies By Phage Display Technology,” Annu. Rev.Immunol. 12.433-455).

The antibodies or protein of interest may be subjected to sequencing byEdman degradation, which is well known to those of skill in the art. Thepeptide information generated from mass spectrometry or Edmandegradation can be used to design probes or primers that are used toclone the protein of interest.

An alternative method of cloning the protein of interest is by “panning”using purified B7-H3 or portions thereof for cells expressing theantibody or protein of interest. B7-H3 exists in a “2Ig” form and as a“4Ig” form. The amino acid sequence of the “2Ig” form of human B7-H3 is(SEQ ID NO: 1):

MLRRRGSPGM GVHVGAALGA LWFCLTGALE VQVPEDPVVALVGTDATLCC SFSPEPGFSL AQLNLIWQLT DTKQLVHSFAEGQDQGSAYA NRTALFPDLL AQGNASLRLQ RVRVADEGSFTCFVSIRDFG SAAVSLQVAA PYSKPSMTLE PNKDLRPGDTVTITCSSYRG YPEAEVFWQD GQGVPLTGNV TTSQMANEQGLFDVHSVLRV VLGANGTYSC LVRNPVLQQD AHGSVTITGQPMTFPPEALW VTVGLSVCLI ALLVALAFVC WRKIKQSCEEENAGAEDQDG EGEGSKTALQ PLKHSDSKED DGQEIA

The cDNA sequence encoding the “2Ig” form of human B7-H3 is (SEQ ID NO:2):

atgctgcgtc ggcggggcag ccctggcatg ggtgtgcatgtgggtgcagc cctgggagca ctgtggttct gcctcacaggagccctggag gtccaggtcc ctgaagaccc agtggtggcactggtgggca ccgatgccac cctgtgctgc tccttctcccctgagcctgg cttcagcctg gcacagctca acctcatctggcagctgaca gataccaaac agctggtgca cagctttgctgagggccagg accagggcag cgcctatgcc aaccgcacggccctcttccc ggacctgctg gcacagggca acgcatccctgaggctgcag cgcgtgcgtg tggcggacga gggcagcttcacctgcttcg tgagcatccg ggatttcggc agcgctgccgtcagcctgca ggtggccgct ccctactcga agcccagcatgaccctggag cccaacaagg acctgcggcc aggggacacggtgaccatca cgtgctccag ctaccggggc taccctgaggctgaggtgtt ctggcaggat gggcagggtg tgcccctgactggcaacgtg accacgtcgc agatggccaa cgagcagggcttgtttgatg tgcacagcgt cctgcgggtg gtgctgggtgcgaatggcac ctacagctgc ctggtgcgca accccgtgctgcagcaggat gcgcacggct ctgtcaccat cacagggcagcctatgacat tccccccaga ggccctgtgg gtgaccgtggggctgtctgt ctgtctcatt gcactgctgg tggccctggctttcgtgtgc tggagaaaga tcaaacagag ctgtgaggaggagaatgcag gagctgagga ccaggatggg gagggagaaggctccaagac agccctgcag cctctgaaac actctgacagcaaagaagat gatggacaag aaatagcc

The amino acid sequence of the “2Ig” form of human B7-H3 (SEQ ID NO: 1)(shown in bold and underline below) is completely embraced within the“4Ig” form of human B7-H3 (SEQ ID NO: 76):

MLRRRGSPGM GVHVGAALGA LWFCLTGAL E VQVPEDPVVALVGTDATLCC SFSPEPGFSL AQLNLIWQLT DTKQLVHSFAEGQDQGSAYA NRTALFPDLL AQGNASLRLQ RVRVADEGSFTCFVSIRDFG SAAVSLQVAA PYSKPSMTLE PNKDLRPGDTVTITCSSYQG YPEAEVFWQD GQGVPLTGNV TTSQMANEQGLFDVHSILRV VLGANGTYSC LVRNPVLQQD AHSSVTITPQ RSPTGAVEVQ VPEDPVVALV GTDATLRCSF SPEPGFSLAQLNLIWQLTDT KQLVHSFTEG RDQGSAYANR TALFPDLLAQGNASLRLQRV RVADEGSFTC FVSIRDFGSA AVSLQVAAPYSKPSMTLEPN KDLRPGDTVT ITCSSYRGYP EAEVFWQDGQGVPLTGNVTT SQMANEQGLF DVHSVLRVVL GANGTYSCLVRNPVLQQDAH GSVTITGQPM TFPPEALWVT VGLSVCLIALLVALAFVCWR KIKQSCEEEN AGAEDQDGEG EGSKTALQPL KHSDSKEDDG QEIA

The cDNA sequence encoding the “4Ig” form of human B7-H3 is (SEQ ID NO:77); residues encoding the “2Ig” form of B&-H3 are shown in bold andunderlined:

atgctgcgtc ggcggggcag ccctggcatg ggtgtgcatgtgggtgcagc cctgggagca ctgtggttct gcctcacagg agccctggag gtccaggtcc ctgaagaccc agtggtggcactggtgggca ccgatgccac cctgtgctgc tccttctcccctgagcctgg cttcagcctg gcacagctca acctcatctggcagctgaca gataccaaac agctggtgca cagctttgctgagggccagg accagggcag cgcctatgcc aaccgcacggccctcttccc ggacctgctg gcacagggca acgcatccctgaggctgcag cgcgtgcgtg tggcggacga gggcagcttcacctgcttcg tgagcatccg ggatttcggc agcgctgccgtcagcctgca ggtggccgct ccctactcga agcccagcatgaccctggag cccaacaagg acctgcggcc aggggacacggtgaccatca cgtgctccag ctaccagggc taccctgaggctgaggtgtt ctggcaggat gggcagggtg tgcccctgactggcaacgtg accacgtcgc agatggccaa cgagcagggcttgtttgatg tgcacagcat cctgcgggtg gtgctgggtgcaaatggcac ctacagctgc ctggtgcgca accccgtgctgcagcaggat gcgcacagct ctgtcaccat cacaccccag agaagcccca caggagccgt g gaggtccag gtccctgagg acccggtggt ggccctagtg ggcaccgatg ccaccctgcgctgctccttc tcccccgagc ctggcttcag cctggcacagctcaacctca tctggcagct gacagacacc aaacagctggtgcacagttt caccgaaggc cgggaccagg gcagcgcctatgccaaccgc acggccctct tcccggacct gctggcacaaggcaatgcat ccctgaggct gcagcgcgtg cgtgtggcggacgagggcag cttcacctgc ttcgtgagca tccgggatttcggcagcgct gccgtcagcc tgcaggtggc cgctccctactcgaagccca gcatgaccct ggagcccaac aaggacctgcggccagggga cacggtgacc atcacgtgct ccagctaccggggctaccct gaggctgagg tgttctggca ggatgggcagggtgtgcccc tgactggcaa cgtgaccacg tcgcagatggccaacgagca gggcttgttt gatgtgcaca gcgtcctgcgggtggtgctg ggtgcgaatg gcacctacag ctgcctggtgcgcaaccccg tgctgcagca ggatgcgcac ggctctgtcaccatcacagg gcagcctatg acattccccc cagaggccctgtgggtgacc gtggggctgt ctgtctgtct cattgcactgctggtggccc tggctttcgt gtgctggaga aagatcaaacagagctgtga ggaggagaat gcaggagctg aggaccaggatggggaggga gaaggctcca agacagccct gcagcctctgaaacactctg acagcaaaga agatgatgga caagaaatag cc

The “panning” procedure may be conducted by obtaining a cDNA libraryfrom tissues or cells that express B7-H3, over-expressing the cDNAs in asecond cell type, and screening the transfected cells of the second celltype for a specific binding to B7-H3. Detailed descriptions of themethods used in cloning mammalian genes coding for cell surface proteinsby “panning” can be found in the art (see, for example, Aruffo, A. etal. (1987) “Molecular Cloning Of A CD28 cDNA By A High-Efficiency COSCell Expression System,” Proc. Natl. Acad. Sci. (U.S.A.) 84:8573-8577and Stephan, J. et al. (1999) “Selective Cloning Of Cell SurfaceProteins Involved In Organ Development: Epithelial Glycoprotein IsInvolved In Normal Epithelial Differentiation,” Endocrinol.140:5841-5854).

cDNAs encoding anti-B7-H3 antibodies, and other B7-H3 peptide agonists,antagonists and modulators can be obtained by reverse transcribing themRNAs from a particular cell type according to standard methods in theart. Specifically, mRNA can be isolated using various lytic enzymes orchemical solutions according to the procedures set forth in Sambrook etal. supra or extracted by commercially available nucleic-acid-bindingresins following the accompanying instructions provided by manufacturers(e.g., Qiagen, Invitrogen, Promega). The synthesized cDNAs are thenintroduced into an expression vector to produce the antibody or proteinof interest in cells of a second type. It is implied that an expressionvector must be replicable in the host cells either as episomes or as anintegral part of the chromosomal DNA. Suitable expression vectorsinclude but are not limited to plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, and cosmids.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

Any host cells capable of over-expressing heterologous DNAs can be usedfor the purpose of isolating the genes encoding the antibody,polypeptide or protein of interest. Non-limiting examples of suitablemammalian host cells include but are not limited to COS, HeLa, and CHOcells. Preferably, the host cells express the cDNAs at a level of about5-fold higher, more preferably 10-fold higher, even more preferably20-fold higher than that of the corresponding endogenous antibody orprotein of interest, if present, in the host cells. Screening the hostcells for a specific binding to B7-H3 is effected by an immunoassay orFACS. A cell over-expressing the antibody or protein of interest can beidentified.

Various techniques are also available which may now be employed toproduce mutant B7-H3 peptide agonists, antagonists, and modulators whichencodes for additions, deletions, or changes in amino acid sequence ofthe resultant protein relative to the parent B7-H3 peptide agonist,antagonist or modulator molecule.

The invention includes polypeptides comprising an amino acid sequence ofthe antibodies of this invention. The polypeptides of this invention canbe made by procedures known in the art. The polypeptides can be producedby proteolytic or other degradation of the antibodies, by recombinantmethods (i.e., single or fusion polypeptides) as described above or bychemical synthesis. Polypeptides of the antibodies, especially shorterpolypeptides up to about 50 amino acids, are conveniently made bychemical synthesis. Methods of chemical synthesis are known in the artand are commercially available. For example, an anti-B7-H3 polypeptidecould be produced by an automated polypeptide synthesizer employing thesolid phase method.

IV. Methods for Screening Polypeptides and Monoclonal Antibodies

Several methods may be used to screen polypeptides and monoclonalantibodies that bind to B7-H3. It is understood that “binding” refers tobiologically or immunologically relevant specific binding, and does notrefer to non-specific binding that may occur, for example, when animmunoglobulin is used at a very high concentration against anon-specific target. In one embodiment, monoclonal antibodies arescreened for binding to B7-H3 using standard screening techniques. Inthis manner, anti-B7-H3 monoclonal antibody was obtained. The preferredhybridomas of the present invention are those that produce antibodiesBRCA69D, BRCA84D or PRCA157.

Additional monoclonal antibodies that bind to B7-H3 may be identified.For this purpose, monoclonal antibodies are screened for theirdifferential ability to bind to cancerous tissues but not tonon-cancerous cells. In one embodiment, monoclonal antibodies which bindto B7-H3 and that are also cross-reactive to human cancerous cells ortissues, but not to normal cells or tissues to the same degree, areselected. One method that may be employed for screening isimmunohistochemistry (IHC). Standard immunohistochemical techniques areknown to those of average skill in the art. See, for example, ANIMALCELL CULTURE METHODS (J. P. Mather and D. Barnes, eds., Academic Press,NY, Vol. 57, Ch. 18 and 19, pp. 314-350, 1998). Biological samples(e.g., tissues) may be obtained from biopsies, autopsies, or necropsies.To ascertain if B7-H3 is present only on cancerous cells, anti-B7-H3antibodies may be used to detect the presence of B7-H3 on tissues fromindividuals with cancer while other non-cancerous tissues from theindividual suffering from cancer or tissues from individuals withoutcancer are used as a control. The tissue can be embedded in a solid orsemi-solid substance that prevents damage during freezing (e.g., agarosegel or OCT) and then sectioned for staining Cancers from differentorgans and at different grades can be used to screen monoclonalantibodies. Examples of tissues that may be used for screening purposesinclude but are not limited to ovary, breast, lung, prostate, colon,kidney, skin, thyroid, brain, heart, liver, stomach, nerve, bloodvessels, bone, upper digestive tract, and pancreas. Examples ofdifferent cancer types that may be used for screening purposes include,but are not limited to, carcinomas, adenocarcinomas, sarcomas,adenosarcomas, lymphomas, and leukemias.

In yet another alternative, cancerous cells lines such as HMEC(BioWhittaker CC-2251), HUVEC (Primary endothelial cells), BT-474(ATCC#HTB-20), MCF7 (ATCC#HTB22), MDA-MB-175-VII (ATCC#HB-25),MDA-MB-361 (ATCC#HB-27), SKBR3 (ATCC#HTB-30), A549 (ATCC#CCL-185),Calu-3 (ATCC#HTB-55), SKMES-I (ATCC#HTB-58), ES-2 (ATCC#CRL-1978), SKOV3(ATCC#HTB-77), Panc-1 (ATCC#CRL-1469), AsPC-I (ATCC#CRL-1682), HPAF-II(ATCC#CRL-1997), Hs700T (ATCC#HTB-174), Colo205 (ATCC#CCL-222), HT-29(ATCC#HTB-38), SW480 (ATCC#CCL-228), SW948 (ATCC#CCL-237), 293 (ATCC#CRL-1573), 786-0 (ATCC#CRL-1932), A498 (ATCC#HTB-44), Caki-2(ATCC#HTB-47), COS-7 (ATCC#CRL-1651), RL-65 (ATCC #CRL-10345), SV-T2(ATCC#CCL-163.1), 22RV1 (ATCC#CRL-2505), DU145 (ATCC#HTB-81), LNCaP(ATCC#CRL-1740), PC-3 (ATCC#CRL-1435), HT29 (ATCC#HTB-38), Hs746T(ATCC#HTB-135), NCI-N87 (ATCC#CRL-5822) and normal cells from theirrespective tissues may be used to screen for monoclonal antibodies whichare specific for cancerous tissue. Primary, or low passage, cellcultures derived from normal tissues from different organs, includingbut not limited to, kidney, ovary, breast, lung, prostate, colon,kidney, skin, thyroid, aortic smooth muscle, and endothelial cells canbe used as negative controls. The cancerous or non-cancerous cells canbe grown on glass slides or coverslips, or on plastic surfaces, orprepared in a CellArray™ device, as described in WO 01/43869, andscreened for the binding of antibody using IHC as described above fortissues. Alternatively, cells may be removed from the growth surfaceusing non-proteolytic means and spun into a pellet, which is thenembedded and treated as tissues for IHC analysis as described above.Cells may be inoculated into immunodeficient animals, a tumor allowed togrow, and then this tumor may be harvested, embedded, and used as atissue source for IHC analysis. In another alternative, single cells maybe screened by incubating with the primary antibody, a secondary“reporter” antibody linked to a fluorescent molecule and then analyzedusing a fluorescent activated cell-sorting (FACS) machine.

Any of several different detection systems may be utilized to detectbinding of antibodies to tissue section. Typically, immunohistochemistryinvolves the binding of a primary antibody to the tissue and then asecondary antibody reactive against the species from the primaryantibody was generated and conjugated to a detectable marker (e.g.,horseradish peroxidase, HRP, or diaminobenzedine, DAB). One alternativemethod that may be used is polyclonal mirror image complementaryantibodies or polyMICA™ (polyclonal Mirror Image ComplementaryAntibodies; The Binding Site Limited, Birmingham, UK; Mangham, D. C. etal. (1999) “A Novel Immunohistochemical Detection System Using MirrorImage Complementary Antibodies (MICA),” Histopathology 35(2):129-33).The PoIyMICA™ technique can be used to test binding of primaryantibodies (e.g., anti-B7-H3 antibodies) to normal and cancerous tissue.Several kinds of polyMICA™ Detection kits are commercially available:Product No. HK004.D is a polyMICA® Detection kit which uses DABchromagen; Product No. HK004.A is a polyMICA® Detection kit which usesAEC chromagen. Alternatively, the primary antibody may be directlylabeled with the detectable marker.

The first step in IHC screening to select for an appropriate antibody isthe binding of primary antibodies raised in mice (e.g., anti-B7-H3antibodies) to one or more immunogens (e.g., cells or tissue samples).In one embodiment, the tissue sample is sections of frozen tissue fromdifferent organs. The cells or tissue samples can be either cancerous ornon-cancerous.

Frozen tissues can be prepared, sectioned, with or without fixation, andIHC performed by any of a number of methods known to one familiar withthe art (see, for example, Stephan et al. (1999) “Distribution AndFunction Of The Adhesion Molecule BEN During Rat Development,” Dev.Biol. 212:264-277 and Stephan et al. (1999) “Selective Cloning Of CellSurface Proteins Involved In Organ Development: Epithelial GlycoproteinIs Involved In Normal Epithelial Differentiation,” Endocrinology140:5841-5854).

V. Methods of Characterizing Anti-B7-H3 Antibodies

Any of several methods can be used to characterize anti-B7-H3antibodies. One method is to identify the epitope to which it binds.Epitope mapping is commercially available from various sources, forexample, Pepscan Systems (Lelystad, The Netherlands). Epitope mappingcan be used to determine the sequence to which an anti-B7-H3 antibodybinds. The epitope can be a linear epitope, i.e., contained in a singlestretch of amino acids, or a conformational epitope formed by athree-dimensional interaction of amino acids that may not necessarily becontained in a single stretch.

Peptides of varying lengths (e.g., preferably at least 4-6 amino acidslong) can be isolated or synthesized {e.g., recombinantly) and used forbinding assays with anti-B7-H3 antibody. The epitope to which anti-B7-H3antibody binds can be determined in a systematic screening by usingoverlapping peptides derived from the extracellular sequence anddetermining binding by anti-B7-H3 antibody.

Yet another method that can be used to characterize an anti-B7-H3antibody is to use competition assays with other antibodies known tobind to the same antigen, i.e., B7-H3 to determine if anti-B7-H3antibodies binds to the same epitope as other antibodies. Examples ofcommercially available antibodies to B7-H3 may be available and may beidentified using the binding assays taught herein. Competition assaysare well known to those of skill in the art, and such procedures andillustrative data are detailed further in the Examples. Anti-B7-H3antibodies can be further characterized by the tissues, type of canceror type of tumor to which they bind.

Another method of characterizing anti-B7-H3 antibodies is by the antigento which it binds. Anti-B7-H3 antibodies were used in Western blots withcell lysates from various human cancers. As is known to one of skill inthe art, Western blotting can involve running cell lysates and/or cellfractions on a denaturing or non-denaturing gel, transferring theproteins to nitrocellulose paper, and then probing the blot with anantibody (e.g., anti-B7-H3 antibody) to see which proteins are bound bythe antibody. B7-H3 is associated with various human cancers ofdifferent tissues including, but not limited to colon, breast, ovary,pancreas and lung.

VI. Methods of Diagnosing Cancer Using Anti-B7-H3 Antibodies and B7-H3Modulators

Monoclonal antibodies to B7-H3 made by the methods disclosed herein maybe used to identify the presence or absence of cancerous cells in avariety of tissues, including but not limited to, ovary, breast, lung,prostate, colon, kidney, pancreas, skin, thyroid, brain, heart, liver,stomach, nerve, blood vessels, bone, and upper digestive tract, forpurposes of diagnosis. Monoclonal antibodies to B7-H3 made by themethods disclosed herein may also be used to identify the presence orabsence of cancerous cells, or the level thereof, which are circulatingin blood after their release from a solid tumor. Such circulatingantigen may be an intact B7-H3 antigen, or a fragment thereof thatretains the ability to be detected according to the methods taughtherein. Such detection may be effected by FACS analysis using standardmethods commonly used in the art.

These uses can involve the formation of a complex between B7-H3 and anantibody that binds specifically to B7-H3. Examples of such antibodiesinclude but are not limited to those anti-B7-H3 monoclonal antibodiesproduced by the hybridomas BRCA84D, BRCA69D, and PRCA157. The formationof such a complex can be in vitro or in vivo. Without being bound bytheory, monoclonal antibody anti-B7-H3 can bind to B7-H3 through theextracellular domain of B7-H3 and may then be internalized.

In a preferred embodiment of the diagnostic methods of this invention,the antibody bears a detectable label. Examples of labels that may beused include a radioactive agent or a fluorophore, such as phycoerythrinor fluorescein isothiocyanate (also known as fluoroisothiocyanate orFITC).

As with other known antibodies used commercially for diagnostic andtherapeutic purposes, the target antigen of this invention is broadlyexpressed in normal tissue. It is also up regulated in some tumors.Therefore, the particular dosages and routes of delivery of theantibodies of this invention as used for diagnostic or therapeuticagents will be tailored to the particular tumor or disease state athand, as well as to the particular individual being treated.

One method of using the antibodies for diagnosis is in vivo tumorimaging by linking the antibody to a radioactive or radio-opaque agent,administering the antibody to the individual and using an x-ray or otherimaging machine to visualize the localization of the labeled antibody atthe surface of cancer cells expressing the antigen. The antibody isadministered at a concentration that promotes binding at physiologicalconditions.

In vitro techniques for detection of B7-H3 are routine in the art andinclude enzyme linked immunosorbent assays (ELISAs),immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA),radioimmunoassay (RIA), and Western blot analysis.

In aspects of this invention, methods of radioimaging of tumors orneoplasms, or of measuring the effectiveness of a method of treatmentwith a radiolabeled antibody, comprising the step of administering aradiolabeled, tumor-specific antibody to an individual following thepractice of this invention. The radiolabeled antibody may be amonoclonal or polyclonal antibody comprising a radiolabel, preferablyselected from the group consisting of Technetium-99m, Indium-111,Iodine-131, Rhenium-186, Rhenium-188, Samarium-153, Lutetium-177,Copper-64, Scandium-47, Yttrium-90. Monoclonal antibodies labeled withtherapeutic radionuclides such as Iodine-131, Rhenium-188, Holmium-166,Samarium-153 and Scandium-47, which do not compromise theimmunoreactivity of antibodies and are not broken down in vivo, areespecially preferred. The person skilled in the art will appreciate thatother radioactive isotopes are known, and may be suitable for specificapplications. The radioimaging may be conducted using Single PhotonEmission Computer Tomography (SPECT), Position Emission Tomography(PET), Computer Tomography (CT) or Magnetic Resonance Imaging (MRI).Correlative imaging, which permits greater anatomical definition oflocation of metastases located by radioimmunoimaging, is alsocontemplated.

In other methods, the cancerous cells are removed and the tissueprepared for immunohistochemistry by methods well known in the art(e.g., embedding in a freezing compound, freezing and sectioning, withor without fixation; fixation and paraffin embedding with or withoutvarious methods of antigen retrieval and counterstaining) The monoclonalantibodies may also be used to identify cancerous cells at differentstages of development. The antibodies may also be used to determinewhich individuals' tumors express the antigen on their surface at apre-determined level and are thus candidates for immunotherapy usingantibodies directed against said antigen. The antibodies may recognizeboth primary and metastasizing cancers that express B7-H3. As usedherein, detection may include qualitative and/or quantitative detectionand may include comparing the level measured to a normal cell for anincreased level of expression of B7-H3 in cancerous cells.

The invention also provides methods of aiding diagnosis of cancercharacterized by cancer cells that express B7-H3 in an individual usingany antibody that binds to B7-H3 and any other methods that can be useddetermine the level of B7-H3 expression. As used herein, methods for“aiding diagnosis” means that these methods assist in making a clinicaldetermination regarding the classification, or nature, of cancer, andmay or may not be conclusive with respect to the definitive diagnosis.Accordingly, a method of aiding diagnosis of cancer can comprise thestep of detecting the level of B7-H3 in a biological sample from theindividual and/or determining the level of B7-H3 expression in thesample. Antibodies recognizing the antigen or a portion thereof may alsobe used to create diagnostic immunoassays for detecting antigen releasedor secreted from living or dying cancer cells in bodily fluids,including but not limited to, blood, saliva, urine, pulmonary fluid, orascites fluid.

Not all cells in a particular tumor of interest will express B7-H3, andcancerous cells in other tissues may express B7-H3, thus an individualshould be screened for the presence or absence of B7-H3 on cancerouscells to determine the usefulness of immunotherapy in the individual.The anti-B7-H3 antibodies made by the methods disclosed herein may beused to determine whether an individual diagnosed with cancer may bedeemed a candidate for immunotherapy using antibodies directed againstB7-H3. In one embodiment, a cancerous tumor or a biopsy sample may betested for expression of B7-H3, using antibodies directed against B7-H3.Individuals with cancer cells that express B7-H3 are suitable candidatesfor immunotherapy using antibodies directed against B7-H3. Staining withanti-B7-H3 antibody may also be used to distinguish cancerous tissuesfrom normal tissues.

Methods of using anti-B7-H3 antibodies for diagnostic purposes areuseful both before and after any form of anti-cancer treatment, e.g.,chemotherapy or radiation therapy, to determine which tumors are mostlikely to respond to a given treatment, prognosis for individual withcancer, tumor subtype or origin of metastatic disease, and progressionof the disease or response to treatment.

The compositions of this invention are also suitable for diagnosis ofdisease states other than cancer, using the methods generally describedabove in application with other diseased (non-cancerous) cells. Diseasestates suitable for use in the methods of this invention include, butare not limited to, diseases or disorders associated with inflammatoryor autoimmune responses in individuals. The methods described above maybe used for modulating inflammatory or autoimmune responses inindividuals. Diseases and conditions resulting from inflammation andautoimmune disorders that may be subject to diagnosis and/or treatmentusing the compositions and methods of the invention include, by way ofillustration and not of limitation, multiple sclerosis, meningitis,encephalitis, stroke, other cerebral traumas, inflammatory bowel diseaseincluding ulcerative colitis and Crohn's disease, myasthenia gravis,lupus, rheumatoid arthritis, asthma, acute juvenile onset diabetes, AIDSdementia, atherosclerosis, nephritis, retinitis, atopic dermatitis,psoriasis, myocardial ischemia and acute leukocyte-mediated lung injury.

Still other indications for diagnostic and/or therapeutic use ofantibodies and other therapeutic agents of the invention includeadministration to individuals at risk of organ or graft rejection. Overrecent years there has been a considerable improvement in the efficiencyof surgical techniques for transplanting tissues and organs such asskin, kidney, liver, heart, lung, pancreas and bone marrow. Perhaps theprincipal outstanding problem is the lack of satisfactory agents forinducing immunotolerance in the recipient to the transplanted allograftor organ. When allogeneic cells or organs are transplanted into a host(i.e., the donor and donee are different individuals from the samespecies), the host immune system is likely to mount an immune responseto foreign antigens in the transplant (host-versus-graft disease)leading to destruction of the transplanted tissue.

Uses described anywhere in this application for anti-B7-H3 antibodiesalso encompass the use of other B7-H3 agonists, antagonists andmodulators as described herein. In such embodiments, the B7-H3 agonist,antagonist or other non-antibody modulator is substituted for the B7-H3antibody in the steps described, and alterations within the scope of theordinarily skilled practitioner are made to tailor the method to thesubstituted B7-H3 modulatory composition.

Monoclonal antibodies to B7-H3 made by the methods disclosed herein maybe used to identify the presence or absence of human cancer stem cellsin a variety of tissues. Cancer stem cells (CSCs) have been hypothesizedto play a role in tumor growth and metastasis (Ghotra, V. P. et al.(2009) “The Cancer Stem Cell Microenvironment And Anti-Cancer Therapy,”Int. J. Radiat. Biol. 85(11):955-962; Gupta, P. B. et al. (2009) “CancerStem Cells: Mirage Or Reality?” Nat. Med. 15(9):1010-1012; Lawson, J. C.et al. (2009) “Cancer Stem Cells In Breast Cancer And Metastasis,”Breast Cancer Res. Treat. 118(2):241-254; Hermann, P. C. et al. (2009)“Pancreatic Cancer Stem Cells Insights And Perspectives,” Expert Opin.Biol. Ther. 9(10):1271-1278; Schatton, T. et al. (2009) “IdentificationAnd Targeting Of Cancer Stem Cells,” Bioessays 31(10):1038-1049; Mittal,S. et al. (2009) “Cancer Stem Cells: The Other Face Of Janus,” Amer. J.Med. Sci. 338(2):107-112; Alison, M. R. et al. (2009) “Stem Cells AndLung Cancer: Future Therapeutic Targets?” Expert Opin. Biol. Ther.9(9):1127-1141; Charafe-Jauffret, E. et al. (2009) “Breast Cancer StemCells: Tools And Models To Rely On,” BMC Cancer 9:202; Scopelliti, A. etal. (2009) “Therapeutic Implications Of Cancer Initiating Cells,” ExpertOpin. Biol. Ther. 9(8):1005-1016; PCT Publication WO 2008/091908). Underthis hypothesis, the CSCs provide a small, distinct subset of cellswithin each tumor that are capable of indefinite self-renewal and ofdeveloping into the more adult tumor cell(s) that are relatively limitedin replication capacity. It has been hypothesized that these cancer stemcells might be more resistant to chemotherapeutic agents, radiation orother toxic conditions, and thus, persist after clinical therapies andlater grow into secondary tumors, metastases or be responsible forrelapse. It has been suggested that CSCs can arise either from ‘normal’tissue stem cells or from more differentiated tissue progenitor cells.

Human cancer stem cells have several defining characteristics. Suchcharacteristics are described in PCT Publication WO 2008/091908 and arehereby incorporated by reference. Monoclonal antibodies to cell surfacetargets on cancer stem cells can be used to identify the presence orabsence of cancer stem cells in a variety of tissues. As indicatedabove, monoclonal antibodies to B7-H3 made by the methods disclosedherein may also be used to identify the presence or absence of cancerstem cells, or the level of cancer stem cells in a sample or tissue orin circulation after their release from a solid tumor. Such circulatingantigen may be an intact B7-H3 antigen, or a fragment thereof thatretains the ability to be detected according to the methods taughtherein. Such detection may be effected by FACS analysis using standardmethods commonly used in the art. In another embodiment, such detectionmay be effected by immunohistochemical analysis of tissue samples usingstandard methods commonly used in the art.

These uses can involve the formation of a complex between B7-H3 and anantibody that binds specifically to B7-H3 on cancer stem cells. Examplesof such antibodies include but are not limited to those anti-B7-H3monoclonal antibodies produced by the hybridomas BRCA84D, BRCA69D, andPRCA157. The formation of such a complex can be in vitro or in vivo.

Uses described in this application that recite their use for anti-B7-H3antibodies also encompass the use of other B7-H3 agonists, antagonistsand modulators as described herein for the use of identification andtreatment of cancer stem cells. In such embodiments, anti-B7-H3antibodies and other B7-H3 agonists, antagonists and modulators are usedfor identification, diagnosis or therapeutic treatment of cancer stemcells using similar methods described, and alterations within the scopeof the ordinary skilled practitioner are made to tailor the method tothe identification/diagnosis or treatment of cancer stem cells.

VII. Preferred Compositions of the Present Invention

The present invention encompasses compositions, including pharmaceuticalcompositions, comprising anti-B7-H3 antibodies, polypeptides derivedfrom anti-B7-H3 antibodies, polynucleotides comprising sequence encodinganti-B7-H3 antibodies, and other agents as described herein. As usedherein, compositions further comprises one or more antibodies,polypeptides and/or proteins that bind to B7-H3, B7-H3 agonists,antagonists, modulators, and/or one or more polynucleotides comprisingsequences encoding one or more antibodies, polypeptides and proteinsthat bind to B7-H3.

The invention further provides for conjugates of any B7-H3 peptideagonist, antagonist or modulator, and additional chemical structuresthat support the intended function or functions of the particular B7-H3peptide agonist, antagonist or modulator.

These conjugates include B7-H3 peptide agonist, antagonist or modulatorcovalently bound to a macromolecule such as any insoluble, solid supportmatrix used in the diagnostic, screening or purification proceduresdiscussed herein. Suitable matrix materials include any substance thatis chemically inert, has high porosity and has large numbers offunctional groups capable of forming covalent linkages with peptideligands. Examples of matrix materials and procedures for preparation ofmatrix-ligand conjugates are described in Dean et al. (Eds) AFFINITYCHROMATOGRAPHY: A PRACTICAL APPROACH, IRL Press (1985); Lowe, “AnIntroduction to Affinity Chromatography”, in Work et al. (eds)LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, Vol. 7,Part II, North-Holland (1979); Porath et al., “Biospecific AffinityChromatography”, in Neurath, H. et al. (eds), THE PROTEINS, 3rd ed.,Vol. 1, pp. 95-178 (1975); and Schott, H. AFFINITY CHROMATOGRAPHY, MacelDekker, Inc. NY (1984).

Also provided herein are conjugates of B7-H3 peptide agonist, antagonistor modulator and any reporter moiety used in the diagnostic proceduresdiscussed herein. The B7-H3 peptide agonist, antagonist or modulatoragents, polypeptides and proteins of this invention, includinganti-B7-H3 antibodies, are further identified and characterized by any(one or more) of the following criteria:

-   -   (a) an ability to specifically bind to B7-H3 (and in particular        B7-H3 molecules that are expressed on the surfaces of cancer        cells, including but not limited to kidney, prostate, or lung,        cancer cells);    -   (b) an ability to competitively inhibits preferential binding of        a known anti-B7-H3 antibody to B7-H3, including the ability to        preferentially bind to the same B7-H3 epitope to which the        original antibody preferentially binds;    -   (c) an ability to bind to a portion of B7-H3 that is exposed on        the surface of a living cell in vitro or in vivo;    -   (d) an ability to bind to a portion of B7-H3 that is exposed on        the surface of living cancer cells that express B7-H3;    -   (e) an ability to deliver a chemotherapeutic agent to cancerous        cells (such as kidney, prostate, or lung cancer cells)        expressing B7-H3 on their surface; and/or    -   (f) an ability to deliver a therapeutic agent or detectable        marker into cancer cells (such as but not limited to prostate        cancer cells) expressing B7-H3 on their surface.

A preferred antibody of the invention will exhibit differential IHCstaining of tumor tissue relative to normal, non-cancerous tissue, andwill moreover be capable of testing in primate (and particularlycynomolgus monkey) models of antibody efficacy. Preferred antibodies ofthe present invention will additionally exhibit desirable levels ofaffinity and antigen specificity. Preferred antibodies of the presentinvention will additionally exhibit desirable levels of immunomodulatoryactivity and cellular internalization.

In some embodiments, the antibody of the invention is an antibody thatis produced by hybridoma BRCA84D, BRCA69D, or PRCA157, or progenythereof. The present invention also encompasses various formulations ofantibodies produced by these deposited hybridomas and equivalentantibodies, chimeric antibodies, single chain (scFv), mutants thereof,fusion proteins comprising an antibody portion, humanized antibodies,and any other modified configuration of any of these or equivalentantibodies that comprises an antigen (B7-H3), recognition site of therequired specificity. The invention also provides human antibodiesdisplaying one or more of the biological characteristics of ananti-B7-H3 family member. The equivalent antibodies of the anti-B7-H3family (including humanized antibodies and human antibodies),polypeptide fragments, and polypeptides comprising any of thesefragments are identified and characterized by any (one or more) of thefive criteria described above. Murine and exemplary humanized variabledomain sequences of an anti-B7-H3 antibody are provided in PCTPublication WO 2008/066691. Such sequences are provided by way ofillustration not limitation, and different sequences as well asfragments and variants of the provided sequences, are encompassed withinthe scope of this invention.

BRCA84D, BRCA69D, and PRCA157 are the preferred B7-H3 antibodies of thepresent invention due to their cleaner normal tissue IHC profiles,stronger tumor/normal IHC differential, moderate to strong binding(BIACORE™)/IHC), cross-reactivity to B7-H3 of cynomolgus monkeys andpotent activity toward universal DART molecules (“UDARTs”) relative tothe other antibodies. In particularly preferred embodiments, theinvention encompasses chimeric and humanized variants of these preferredantibodies, as well as native and chimeric and humanized variants ofthese preferred antibodies that possess modified Fc regions as describedbelow. The invention additionally encompasses DART molecules thatpossess the epitope binding regions of such antibodies, particularly inconcert with epitope binding region(s) that bind to the T-cell receptor,NKG2D receptor, or to a tumor-associated antigen or to a hapten such asfluorescein (e.g., fluorescein isothiocyanate (also known asfluoroisothiocyanate or FITC).

In some embodiments, the antibodies, polypeptides and proteins of theinvention that bind to B7-H3 are antibodies, polypeptides and proteinsthat competitively inhibit preferential binding of a herein-specifiedanti-B7-H3 antibody to B7-H3. In some embodiments, the antibodies, thepolypeptides and the proteins preferentially bind to the same epitope onB7-H3 as the antibody mu-anti-B7-H3 preferentially binds.

Accordingly, the invention provides any of the following (orcompositions, including pharmaceutical compositions, comprising any ofthe following): (a) an antibody produced by the host cell with a depositnumber identified above or its progeny; (b) a humanized form of such anantibody; (c) an antibody comprising one or more of the light chainand/or heavy chain variable regions of such an antibody; (d) a chimericantibody comprising variable regions homologous or derived from variableregions of a heavy chain and a light chain of such an antibody, andconstant regions homologous or derived from constant regions of a heavychain and a light chain of a human antibody; (e) an antibody comprisingone or more of the light chain and/or heavy chain CDRs (at least one,two, three, four, five, or six) of such an antibody; (f) an antibodycomprising a heavy and/or a light chain of such an antibody; (g) a humanantibody that is equivalent to such an antibody. A humanized form of theantibody may or may not have CDRs identical to that original antibody,or antibody produced by a host cell with a deposit number identifiedabove. Determination of CDR regions is well within the skill of the art.In some embodiments, the invention provides an antibody which comprisesat least one CDR that is substantially homologous to at least one CDR,at least two, at least three, at least four, at least 5 CDRs of anantibody produced by one of the above-identified deposited hybridomas(or, in some embodiments substantially homologous to all 6 CDRs of oneof these antibodies, or derived from one of these antibodies), orantibody produced by the host cell with a deposit number identifiedabove. Other embodiments include antibodies that have at least two,three, four, five, or six CDR(s) that are substantially homologous to atleast two, three, four, five or six CDRs of an antibody produced from ahybridoma deposited as identified herein, or derived from such anantibody. It is understood that, for purposes of this invention, bindingspecificity and/or overall activity (which may be in terms of deliveringa chemotherapeutic agent to or into cancerous cells to reduce the growthand/or proliferation of cancer cells, to induce apoptotic cell death inthe cancer cell, to delay the development of metastasis, and/or treatingpalliatively) is generally retained, although the extent of activity mayvary compared to an antibody produced by a deposited hybridoma (may begreater or lesser). The invention also provides methods of making any ofthese antibodies. Methods of making antibodies are known in the art andare described herein.

The invention also provides polypeptides comprising an amino acidsequence of the antibodies of the invention. In some embodiments, thepolypeptide comprises one or more of the light chain and/or heavy chainvariable regions of the antibody. In some embodiments, the polypeptidecomprises one or more of the light chain and/or heavy chain CDRs of theantibody. In some embodiments, the polypeptide comprises three CDRs ofthe light chain and/or heavy chain of the antibody. In some embodiments,the polypeptide comprises an amino acid sequence of the antibody thathas any of the following: at least 5 contiguous amino acids of asequence of the original antibody, at least 8 contiguous amino acids, atleast about 10 contiguous amino acids, at least about 15 contiguousamino acids, at least about 20 contiguous amino acids, at least about 25contiguous amino acids, at least about 30 contiguous amino acids,wherein at least 3 of the amino acids are from a variable region of theantibody. In one embodiment, the variable region is from a light chainof the original antibody. In another embodiment, the variable region isfrom a heavy chain of the antibody. In another embodiment, the 5 (ormore) contiguous amino acids are from a complementarity-determiningregion (CDR) of the antibody.

In some embodiments of this invention, cells of this invention thatexpress B7-H3, a portion of B7-H3, anti-B7-H3 antibodies or otherB7-H3-binding polypeptides of this invention are administered directlyto an individual to modulate in vivo B7-H3 biological activity.

The preferred anti-B7-H3 antibodies of the present invention areBRCA84D, BRCA69D and PRCA157, all of which antibodies are murineantibodies reactive toward the human B7-H3 molecule. The amino acid andencoding polynucleotide sequences of the variable light chain andvariable heavy chain of BRCA84D, BRCA69D and PRCA157 are shown belowalong with the respective CDR₁, CDR₂ and CDR₃ domains of each suchchain. Those of skill in the art will therefore be able to constructantibodies having such CDRs, as well as derivatives thereof, capable ofbinding to the epitopes recognized by BRCA84D, BRCA69D and PRCA157.

A. Sequences of BRCA84D

(1) BRCA84D Light Chain Sequences

Amino Acid Sequence of BRCA84D Variable Light Chain (SEQ ID NO: 3):DIAMTQSQKF MSTSVGDRVS VTCKASQNVD TNVAWYQQKPGQSPKALIYS ASYRYSGVPD RFTGSGSGTD FTLTINNVQSEDLAEYFCQQ YNNYPFTFGS GTKLEIKPolynucleotide Sequence Encoding BRCA84D VariableLight Chain (SEQ ID NO: 4): gacattgcga tgacccagtc tcaaaaattc atgtccacatcagtaggaga cagggtcagc gtcacctgca aggccagtcagaatgtggat actaatgtag cctggtatca acagaaaccagggcaatctc ctaaagcact gatttactcg gcatcctaccggtacagtgg agtccctgat cgcttcacag gcagtggatctgggacagat ttcactctca ccatcaacaa tgtgcagtctgaagacttgg cagagtattt ctgtcagcaa tataacaactatccattcac gttcggctcg gggacaaagt tggaaataaa aBRCA84D Variable Light Chain CDR₁ (SEQ ID NO: 5): KASQNVDTNVAPolynucleotide Sequence Encoding BRCA84D VariableLight Chain CDR₁ (SEQ ID NO: 6): aaggccagtc agaatgtgga tactaatgta gccBRCA84D Variable Light Chain CDR₂ (SEQ ID NO: 7): SASYRYSPolynucleotide Sequence Encoding BRCA84D VariableLight Chain CDR₂ (SEQ ID NO: 8): tcggcatcct accggtacag tBRCA84D Variable Light Chain CDR₃ (SEQ ID NO: 9): QQYNNYPFTPolynucleotide Sequence Encoding BRCA84D VariableLight Chain CDR₃ (SEQ ID NO: 10): cagcaatata acaactatcc attcacg

(2) BRCA84D Heavy Chain Sequences

Amino Acid Sequence of BRCA84D Variable Heavy Chain (SEQ ID NO: 11):DVQLVESGGG LVQPGGSRKL SCAASGFTFS SFGMHWVRQAPEKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDNPKNTLFLQMTSLRSED TAMYYCGRGR ENIYYGSRLD YWGQGTTLTV SSPolynucleotide Sequence Encoding BRCA84D VariableHeavy Chain (SEQ ID NO: 12): gatgtgcagc tggtggagtc tgggggaggc ttagtgcagcctggagggtc ccggaaactc tcctgtgcag cctctggattcactttcagt agctttggaa tgcactgggt tcgtcaggctccagagaagg ggctggagtg ggtcgcatac attagtagtgacagtagtgc catctactat gcagacacag tgaagggccgattcaccatc tccagagaca atcccaagaa caccctgttcctgcaaatga ccagtctaag gtctgaggac acggccatgtattactgtgg aagagggagg gaaaacattt actacggtagtaggcttgac tactggggcc aaggcaccac tctcacagtc tcctcaBRCA84D Variable Heavy Chain CDR₁ (SEQ ID NO: 13): FGMHPolynucleotide Sequence Encoding BRCA84D VariableHeavy Chain CDR₁ (SEQ ID NO: 14): tttggaatgcacBRCA84D Variable Heavy Chain CDR₂ (SEQ ID NO: 15): YISSDSSAIYYADTVKPolynucleotide Sequence Encoding BRCA84D VariableHeavy Chain CDR₂ (SEQ ID NO: 16):tacattagta gtgacagtag tgccatctac tatgcagaca cagtgaagBRCA84D Variable Heavy Chain CDR₃ (SEQ ID NO: 17): GRENIYYGSRLDYPolynucleotide Sequence Encoding BRCA84D VariableHeavy Chain CDR₃ (SEQ ID NO: 18):gggagggaaa acatttacta cggtagtagg cttgactac

B. Sequences of BRCA69D

(1) BRCA69D Light Chain Sequences

Amino Acid Sequence of BRCA69D Variable Light Chain (SEQ ID NO: 19):DIQMTQTTSS LSASLGDRVT ISCRASQDIS NYLNWYQQKPDGTVKLLIYY TSRLHSGVPS RFSGSGSGTD YSLTIDNLEQEDIATYFCQQ GNTLPPTFGG GTKLEIKPolynucleotide Sequence Encoding BRCA69D VariableLight Chain (SEQ ID NO: 20): gatatccaga tgacacagac tacatcctcc ctgtctgcctctctgggaga cagagtcacc atcagttgca gggcaagtcaggacattagt aattatttaa actggtatca gcagaaaccagatggaactg ttaaactcct gatctactac acatcacgattacactcagg agtcccatca aggttcagtg gcagtgggtctggaacagat tattctctca ccattgacaa cctggagcaagaagatattg ccacttactt ttgccaacag ggtaatacgcttcctccgac gttcggtgga ggcaccaaac tggaaatcaa aBRCA69D Variable Light Chain CDR₁ (SEQ ID NO: 21): RASQDISNYLNPolynucleotide Sequence Encoding BRCA69D VariableLight Chain CDR₁ (SEQ ID NO: 22): agggcaagtc aggacattag taattattta aacBRCA69D Variable Light Chain CDR₂ (SEQ ID NO: 23): YTSRLHSPolynucleotide Sequence Encoding BRCA69D VariableLight Chain CDR₂ (SEQ ID NO: 14): tacacatcac gattacactc aBRCA69D Variable Light Chain CDR₃ (SEQ ID NO: 25): QQGNTLPPTPolynucleotide Sequence Encoding BRCA69D VariableLight Chain CDR₃ (SEQ ID NO: 15): caacagggta atacgcttcc tccgacg

(2) BRCA69D Heavy Chain Sequences

Amino Acid Sequence of BRCA69D Variable Heavy Chain (SEQ ID NO: 27):QVQLQQSGAE LARPGASVKL SCKASGYTFT SYWMQWVKQRPGQGLEWIGT IYPGDGDTRY TQKFKGKATL TADKSSSTAYMQLSSLASED SAVYYCARRG IPRLWYFDVW GAGTTVTVSSPolynucleotide Sequence Encoding BRCA69D VariableHeavy Chain (SEQ ID NO: 28): caggttcagc tccagcagtc tggggctgag ctggcaagacctggggcttc agtgaagttg tcctgcaagg cttctggctacacctttact agctactgga tgcagtgggt aaaacagaggcctggacagg gtctggaatg gattgggact atttatcctggagatggtga tactaggtac actcagaagt tcaagggcaaggccacattg actgcagata aatcctccag cacagcctacatgcaactca gcagcttggc atctgaggac tctgcggtctattactgtgc aagaagaggg attccacggc tttggtacttcgatgtctgg ggcgcaggga ccacggtcac cgtctcctcaBRCA69D Variable Heavy Chain CDR₁ (SEQ ID NO: 29): SYWMQPolynucleotide Sequence Encoding BRCA69D VariableHeavy Chain CDR₁ (SEQ ID NO: 30): agctactgga tgcagBRCA69D Variable Heavy Chain CDR₂ (SEQ ID NO: 31): TIYPGDGDTR YTQKFKGPolynucleotide Sequence Encoding BRCA69D VariableHeavy Chain CDR₂ (SEQ ID NO: 32):actatttatc ctggagatgg tgatactagg tacactcag aagttcaagg gcBRCA69D Variable Heavy Chain CDR₃ (SEQ ID NO: 33): RGIPRLWYFD VPolynucleotide Sequence Encoding BRCA69D VariableHeavy Chain CDR₃ (SEQ ID NO: 34): agagggattc cacggctttg gtacttcgat gtc

C. Sequences of PRCA157

(1) PRCA157 Light Chain Sequences

Amino Acid Sequence of PRCA157 Variable Light Chain (SEQ ID NO: 35):DIQMTQSPAS LSVSVGETVT ITCRASESIY SYLAWYQQKQGKSPQLLVYN TKTLPEGVPS RFSGSGSGTQ FSLKINSLQPEDFGRYYCQH HYGTPPWTFG GGTNLEIKPolynucleotide Sequence Encoding PRCA157 VariableLight Chain (SEQ ID NO: 36): gacatccaga tgactcagtc tccagcctcc ctatctgtatctgtgggaga aactgtcacc attacatgtc gagcaagtgagagtatttac agttatttag catggtatca gcagaaacagggaaaatctc ctcagctcct ggtctataat acaaaaaccttaccagaggg tgtgccatca aggttcagtg gcagtggatcaggcacacag ttttctctga agatcaacag cctgcagcctgaagattttg ggagatatta ctgtcaacat cattatggtactcctccgtg gacgttcggt ggaggcacca acctggaaat caaaPRCA157 Variable Light Chain CDR₁ (SEQ ID NO: 37): RASESIYSYLAPolynucleotide Sequence Encoding PRCA157 VariableLight Chain CDR₁ (SEQ ID NO: 38): cgagcaagtg agagtattta cagttattta gcaPRCA157 Variable Light Chain CDR₂ (SEQ ID NO: 39): NTKTLPEPolynucleotide Sequence Encoding PRCA157 VariableLight Chain CDR₂ (SEQ ID NO: 40): aatacaaaaa ccttaccaga gPRCA157 Variable Light Chain CDR₃ (SEQ ID NO: 41): QHHYGTPPWPolynucleotide Sequence Encoding PRCA157 VariableLight Chain CDR₃ (SEQ ID NO: 42): caacatcatt atggtactcc tccgtgg

(2) PRCA157 Heavy Chain Sequences

Amino Acid Sequence of PRCA157 Variable Heavy Chain (SEQ ID NO: 43):EVQQVESGGD LVKPGGSLKL SCAASGFTFS SYGMSWVRQTPDKRLEWVAT INSGGSNTYY PDSLKGRFTI SRDNAKNTLYLQMRSLKSED TAMYYCARHD GGAMDYWGQG TSVTVSSPolynucleotideSequence Encoding PRCA157 VariableHeavy Chain (SEQ ID NO: 44): gaggtgcagc aggtggagtc ggggggagac ttagtgaagcctggagggtc cctgaaactc tcctgtgcag cctctggattcactttcagt tcctatggca tgtcttgggt tcgccagactccagacaaga ggctggagtg ggtcgcaacc attaatagtggtggaagtaa cacctactat ccagacagtt tgaaggggcgattcaccatc tccagagaca atgccaagaa caccctttacctgcaaatgc gcagtctgaa gtctgaggac acagccatgtattactgtgc aagacatgac gggggagcta tggactactggggtcaagga acctcagtca ccgtctcctc aPRCA157 Variable Heavy Chain CDR₁ (SEQ ID NO: 45): SYGMSPolynucleotide Sequence Encoding PRCA157 VariableHeavy Chain CDR₁ (SEQ ID NO: 46): tcctatggca tgtctPRCA157 Variable Heavy Chain CDR₂ (SEQ ID NO: 47): VATINSGGSN TYYPDSLKGPolynucleotide Sequence Encoding PRCA157 VariableHeavy Chain CDR₂ (SEQ ID NO: 48):gtcgcaacca ttaatagtgg tggaagtaac acctactatc cagacagttt gaaggggPRCA157 Variable Heavy Chain CDR₃ (SEQ ID NO: 49): HDGGAMDYPolynucleotide Sequence Encoding PRCA157 VariableHeavy Chain CDR₃ (SEQ ID NO: 50): catgacgggg gagctatgga ctac

D. Fc-Engineered B7-H3 Antibodies

In traditional immune function, the interaction of antibody-antigencomplexes with cells of the immune system results in a wide array ofresponses, ranging from effector functions such as antibody-dependentcytotoxicity, mast cell degranulation, and phagocytosis toimmunomodulatory signals such as regulating lymphocyte proliferation andantibody secretion. All of these interactions are initiated through thebinding of the Fc domain of antibodies or immune complexes tospecialized cell surface receptors on hematopoietic cells. The diversityof cellular responses triggered by antibodies and immune complexesresults from the structural heterogeneity of the three Fc receptors:FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI (CD64), FcγRIIA(CD32A) and FcγRIII (CD16) are activating (i.e., immune systemenhancing) receptors; FcγRIIB (CD32B) is an inhibiting (i.e., immunesystem dampening) receptor. The amino acid sequence of the IgG1 Fcregion is shown below (as SEQ ID NO: 51, numbered according to Kabat etal., SEQUENCE OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5^(th) Ed. PublicHealth Service, NIH, MD (1991), expressly incorporated herein byreference, and hereafter referred to as “Kabat EU”):

SEQ ID NO: 51 PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE230        240        250        260DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL270        280        290        300HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY310        320        330        340TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN350        360        370        380NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH390        400        410        420 EALHNHYTQK SLSLSPGK 430        440Residues 230-341 are the Fc CH2 region. Residues 342-447 are the Fc CH3region.

The present invention includes antibodies that specifically bind toB7-H3 that comprise a variant Fc region having one or more amino acidmodifications (e.g., substitutions, deletions, insertions) in one ormore portions, which modifications increase the affinity and avidity ofthe variant Fc region for an FcγR (including activating and inhibitoryFcγRs). In some embodiments, said one or more amino acid modificationsincrease the affinity of the variant Fc region for FcγRIIIA and/orFcγRIIA. In another embodiment, the variant Fc region furtherspecifically binds FcγRIIB with a lower affinity than does the Fc regionof the comparable parent antibody (i.e., an antibody having the sameamino acid sequence as the antibody of the invention except for the oneor more amino acid modifications in the Fc region). In some embodiments,such modifications increase the affinity of the variant Fc region forFcγRIIIA and/or FcγRIIA and also enhance the affinity of the variant Fcregion for FcγRIIB relative to the parent antibody. In otherembodiments, said one or more amino acid modifications increase theaffinity of the variant Fc region for FcγRIIIA and/or FcγRIIA but do notalter the affinity of the variant Fc regions for FcγRIIB relative to theFc region of the parent antibody. In another embodiment, said one ormore amino acid modifications enhance the affinity of the variant Fcregion for FcγRIIIA and FcγRIIA but reduce the affinity for FcγRIIBrelative to the parent antibody. Increased affinity and/or avidityresults in detectable binding to the FcγR or FcγR-related activity incells that express low levels of the FcγR when binding activity of theparent molecule (without the modified Fc region) cannot be detected inthe cells. In other embodiments, the modified molecule exhibitsdetectable binding in cells which express non-FcγR receptor targetantigens at a density of 30,000 to 20,000 molecules/cell, at a densityof 20,000 to 10,000 molecules/cell, at a density of 10,000 to 5,000molecules/cell, at a density of 5,000 to 1,000 molecules/cell, at adensity of 1,000 to 200 molecules/cell or at a density of 200molecules/cell or less (but at least 10, 50, 100 or 150 molecules/cell).

In another embodiment, said one or more modifications to the amino acidsof the Fc region reduce the affinity and avidity of the antibody for oneor more FcγR receptors. In a specific embodiment, the inventionencompasses antibodies comprising a variant Fc region, wherein saidvariant Fc region comprises at least one amino acid modificationrelative to a wild type Fc region, which variant Fc region only bindsone FcγR, wherein said FcγR is FcγRIIIA. In another specific embodiment,the invention encompasses antibodies comprising a variant Fc region,wherein said variant Fc region comprises at least one amino acidmodification relative to a wild type Fc region, which variant Fc regiononly binds one FcγR, wherein said FcγR is FcγRIIA.

Preferably, the binding properties of the molecules of the invention arecharacterized by in vitro functional assays for determining one or moreFcγR mediator effector cell functions (See Section 5.2.7). Theaffinities and binding properties of the molecules, e.g., antibodies, ofthe invention for an FcγR can be determined using in vitro assays(biochemical or immunological based assays) known in the art fordetermining antibody-antigen or Fc-FcγR interactions, i.e., specificbinding of an antigen to an antibody or specific binding of an Fc regionto an FcγR, respectively, including but not limited to ELISA assay,surface plasmon resonance assay, immunoprecipitation assays. In mostpreferred embodiments, the molecules of the invention have similarbinding properties in in vivo models (such as those described anddisclosed herein) as those in in vitro based assays. However, thepresent invention does not exclude molecules of the invention that donot exhibit the desired phenotype in in vitro based assays but doexhibit the desired phenotype in vivo.

In some embodiments, the molecules of the invention comprising a variantFc region comprise at least one amino acid modification (for example,possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications)in the CH3 domain of the Fc region, which is defined as extending fromamino acids 342-447. In other embodiments, the molecules of theinvention comprising a variant Fc region comprise at least one aminoacid modification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, ormore amino acid modifications) in the CH2 domain of the Fc region, whichis defined as extending from amino acids 231-341. In some embodiments,the molecules of the invention comprise at least two amino acidmodifications (for example, possessing 2, 3, 4, 5, 6, 7, 8, 9, or moreamino acid modifications), wherein at least one such modification is inthe CH3 region and at least one such modification is in the CH2 region.The invention further encompasses amino acid modification in the hingeregion. In a particular embodiment, the invention encompasses amino acidmodification in the CH1 domain of the Fc region, which is defined asextending from amino acids 216-230.

In particularly preferred embodiments, the invention encompassesmolecules comprising a variant Fc region wherein said variant confers orhas an increased ADCC activity and/or an increased binding to FcγRIIA(CD32A), as measured using methods known to one skilled in the art andexemplified herein. The ADCC assays used in accordance with the methodsof the invention may be NK dependent or macrophage dependent.

In particularly preferred embodiments, the invention encompassesmolecules comprising a variant Fc region wherein said variant confers orhas an increased ADCC activity and/or an increased binding to FcγRIIIA(CD16A), as measured using methods known to one skilled in the art andexemplified herein. The ADCC assays used in accordance with the methodsof the invention may be NK dependent or macrophage dependent.

The Fc variants of the present invention may be combined with other Fcmodifications, such as those disclosed in U.S. Pat. Nos. 7,632,497;7,521,542; 7,425,619; 7,416,727; 7,371,826; 7,355,008; 7,335,742;7,332,581; 7,183,387; 7,122,637; and 6,737,056; in PCT Publications Nos.WO 2008/105886; WO 2008/002933; WO 2007/021841; WO 2007/106707; WO06/088494; WO 05/115452; WO 05/110474; WO 04/1032269; and in WO04/063351; and in Presta, L. G. et al. (2002) “Engineering therapeuticantibodies for improved function,” Biochem. Soc. Trans. 30(4):487-490;Shields, R. L. et al. (2002) “Lack of fucose on human IgG1 N-linkedoligosaccharide improves binding to human Fcgamma RIII andantibody-dependent cellular toxicity,” J. Biol. Chem. 26;277(30):26733-26740 and Shields, R. L. et al. (2001) “High resolutionmapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII,Fc gamma RIII, and FcRn and design of IgG1 variants with improvedbinding to the Fc gamma R,” J. Biol. Chem. 276(9):6591-6604). Theinvention encompasses combining an Fc variant of the invention withother Fc modifications to provide additive, synergistic, or novelproperties to the modified antibody. Preferably, the Fc variants of theinvention enhance the phenotype of the modification with which they arecombined. For example, if an Fc variant of the invention is combinedwith a mutant known to bind FcγRIIIA with a higher affinity than acomparable wild type Fc region; the combination with a mutant of theinvention results in a greater fold enhancement in FcγRIIIA affinity.

The invention encompasses antibodies that specifically bind to B7-H3which comprise a variant Fc region, wherein the variant Fc regioncomprises at least one amino acid modification (for example, possessing1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative toa wild-type Fc region, such that the molecule has an enhanced effectorfunction relative to a molecule comprising a wild-type Fc region,provided that the variant Fc region does not have or is not solely asubstitution at any one or more of positions 243, 255, 256, 258, 267,268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293,294, 295, 296, 298, 300, 301, 303, 305, 307, 309, 312, 320, 322, 326,329, 330, 332, 331, 333, 334, 335, 337, 338, 339, 340, 359, 360, 373,376, 416, 419, 430, 434, 435, 437, 438, 439. In a specific embodiment,the invention encompasses such antibodies comprising a variant Fcregion, wherein the variant Fc region comprises at least one amino acidmodification (for example, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or moreamino acid modifications) relative to a wild-type Fc region, such thatthe molecule binds an FcγR with an altered affinity relative to amolecule comprising a wild-type Fc region, provided that the variant Fcregion does not have or is not solely a substitution at any one or moreof positions 243, 255, 258, 267, 269, 270, 276, 278, 280, 283, 285, 289,292, 293, 294, 295, 296, 300, 303, 305, 307, 309, 320, 322, 329, 332,331, 337, 338, 340, 373, 376, 416, 419, 434, 435, 437, 438, 439 and doesnot have an alanine at any of positions 256, 290, 298, 312, 326, 333,334, 359, 360, or 430; an asparagine at position 268; a glutamine atposition 272; a glutamine, serine, or aspartic acid at position 286; aserine at position 290; a methionine at position 301; a methionine,glutamine, glutamic acid, or arginine at position 320; a glutamic acidat position 322; an asparagine, serine, glutamic acid, or aspartic acidat position 326; a lysine at position 330; a glutamine at position 334;a glutamic acid at position 334; a methionine at position 334; ahistidine at position 334; a valine at position 334; a leucine atposition 334; a glutamine at position 335; a lysine at position 335; ora threonine at position 339.

The invention also encompasses antibodies that specifically bind toB7-H3 which comprise a variant Fc region, wherein the variant Fc regioncomprises such antibodies comprising a variant Fc region, wherein thevariant Fc region does not have or is not solely a substitution at anyone or more of positions 268, 269, 270, 272, 276, 278, 283, 285, 286,289, 292, 293, 301, 303, 305, 307, 309, 320, 331, 333, 334, 335, 337,338, 340, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439 anddoes not have a histidine, glutamine, or tyrosine at position 280; aserine, glycine, threonine or tyrosine at position 290, an asparagine atposition 294, a lysine at position 295; a proline at position 296; aproline, asparagine, aspartic acid, or valine at position 298; or aleucine or isoleucine at position 300. In another embodiment, theinvention encompasses such antibodies comprising a variant Fc region,wherein the variant Fc region comprises at least one amino acidmodification relative to a wild-type Fc region, such that the moleculebinds an FcγR with a reduced affinity relative to molecule comprising awild-type Fc region provided that the variant Fc region does not have oris not solely a substitution at any one or more of positions 243, 252,254, 265, 268, 269, 270, 278, 289, 292, 293, 294, 295, 296, 298, 300,301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388,389, 414, 416, 419, 434, 435, 437, 438, or 439. In yet anotherembodiment, the invention encompasses such antibodies comprising avariant Fc region, wherein the variant Fc region comprises at least oneamino acid modification relative to a wild-type Fc region, such that themolecule binds an FcγR with an enhanced affinity relative to a moleculecomprising a wild-type Fc region provided that the variant Fc regiondoes not have or is not solely a substitution at any one or more ofpositions 280, 283, 285, 286, 290, 294, 295, 298, 300, 301, 305, 307,309, 312, 315, 331, 333, 334, 337, 340, 360, 378, 398, or 430.

The invention also encompasses antibodies that specifically bind toB7-H3 which comprise a variant Fc region, wherein the variant Fc regiondoes not include or are not solely a substitution at any one or more ofpositions 330, 243, 247, 298, 241, 240, 244, 263, 262, 235, 269, or 328and does not have a leucine at position 243, an asparagine at position298, a leucine at position 241, and isoleucine or an alanine at position240, a histidine at position 244, a valine at position 330, or anisoleucine at position 328.

The invention particularly encompasses antibodies that specifically bindto B7-H3 which comprise a variant Fc region with enhanced effectorfunction and/or altered affinities for activating and/or inhibitoryreceptors, wherein the variant Fc region comprises: (a) any 1, 2, 3, 4,5, or 6 of the following substitutions: S239D, S298A, A330L, I332E,E333A, or K334A; or (b) any of the combinations of substitutions: (1)S298A, E333A, and K334A; (2) S239D and I332E; or (3) S239D, A330L andI332E.

The invention particularly encompasses antibodies that specifically bindto B7-H3 which comprise a variant Fc region with enhanced effectorfunction and/or altered affinities for activating and/or inhibitoryreceptors, wherein the variant Fc region comprises a substitution:

-   (1) at position 288 with asparagine, at position 330 with serine and    at position 396 with leucine;-   (2) at position 334 with glutamic acid, at position 359 with    asparagine, and at position 366 with serine;-   (3) at position 316 with aspartic acid, at position 378 with valine,    and at position 399 with glutamic acid;-   (4) at position 247 with leucine, and a substitution at position 421    with lysine;-   (5) at position 392 with threonine, and at position 396 with    leucine;-   (6) at position 221 with glutamic acid, at position 270 with    glutamic acid, at position 308 with alanine, at position 311 with    histidine, at position 396 with leucine, and at position 402 with    aspartic acid;-   (7) at position 419 with histidine, and a substitution at position    396 with leucine;-   (8) at position 240 with alanine, and at position 396 with leucine;-   (9) at position 410 with histidine, and at position 396 with    leucine;-   (10) at position 243 with leucine, at position 305 with isoleucine,    at position 378 with aspartic acid, at position 404 with serine, and    at position 396 with leucine;-   (11) at position 255 with isoleucine, and at position 396 with    leucine;-   (12) at position 370 with glutamic acid and at position 396 with    leucine;-   (13) at position 270 with glutamic acid;-   or-   (14) any combination of the foregoing (1)-(12) substitutions.

In a specific embodiment, the invention encompasses an antibody thatspecifically binds B7-H3 that comprises a variant Fc region whichcomprises the substitution: F243L, R292P, and Y300L. In a furtherspecific embodiment, the invention encompasses an antibody thatspecifically binds B7-H3 that comprises a variant Fc region whichcomprises the substitution: L235V, F243L, R292P, Y300L, and P396L. Inyet a further specific embodiment, the invention encompasses an antibodythat specifically binds B7-H3 that comprises a variant Fc region whichcomprises the substitution F243L, R292P, Y300L, V305I, and P396L.

In a further specific embodiment, the invention encompasses an antibodythat specifically binds B7-H3 that comprises a variant Fc region whichcomprises a substitution at position 396 with leucine, at position 270with glutamic acid and at position 243 with leucine. In another specificembodiment the molecule further comprises one or more amino acidmodification such as those disclosed herein.

The invention particularly encompasses antibodies that specifically bindto B7-H3 which comprise a variant Fc region with enhanced effectorfunction and/or altered affinities for activating and/or inhibitoryreceptors, that have an amino acid modification at one or more of thefollowing positions: 119, 125, 132, 133, 141, 142, 147, 149, 162, 166,185, 192, 202, 205, 210, 214, 215, 216, 217, 218, 219, 221, 222, 223,224, 225, 227, 229, 231, 232, 233, 235, 240, 241, 242, 243, 244, 246,247, 248, 250, 251, 252, 253, 254, 255, 256, 258, 261, 262, 263, 268,269, 270, 272, 274, 275, 276, 279, 280, 281, 282, 284, 287, 288, 289,290, 291, 292, 293, 295, 298, 301, 303, 304, 305, 306, 307, 308, 309,310, 311, 312, 313, 315, 316, 317, 318, 319, 320, 323, 326, 327, 328,330, 333, 334, 335, 337, 339, 340, 343, 344, 345, 347, 348, 352, 353,354, 355, 358, 359, 360, 361, 362, 365, 366, 367, 369, 370, 371, 372,375, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 404, 406,407, 408, 409, 410, 411, 412, 414, 415, 416, 417, 419, 420, 421, 422,423, 424, 427, 428, 431, 433, 435, 436, 438, 440, 441, 442, 443, 446, or447. Preferably such mutations result in molecules that have beenconferred an effector cell mediated function and, optionally, have analtered affinity for an FcγR as determined using methods disclosed andexemplified herein and known to one skilled in the art.

The invention particularly encompasses antibodies that specifically bindto B7-H3 which comprise a variant Fc region with altered effectorfunction and/or altered affinities for activating and/or inhibitoryreceptors, that have:

-   (I) an amino acid modification at one or more of the following    positions: 235, 240, 241, 243, 244, 247, 262, 263, 269, 298, 328, or    330 and more preferably one or more of the following modifications:    V240A, V240I, F241L, F243L, P244H, S298N, L328I, A330V; wherein such    antibodies exhibit altered effector function relative to antibodies    having a wild-type Fc region that lacks such modification;-   (II) an amino acid modification at one or more of the following    positions: 268, 269, 270, 272, 276, 278, 283, 285, 286, 289, 292,    293, 301, 303, 305, 307, 309, 331, 333, 334, 335, 337, 338, 340,    360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439 and more    preferably one or more of the following modifications: D280H, D280Q,    D280Y, K290G, K290S, K290T, K290Y, E294N, Q295K, Y296P, S298D,    S298N, S298P, S298V, Y300I, Y300L; wherein such antibodies exhibit    altered effector function relative to antibodies having a wild-type    Fc region that lacks such modification;-   (III) an amino acid modification at one or more of the following    positions: 255, 256, 258, 267, 268, 269, 270, 272, 276, 278, 280,    283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301,    303, 305, 307, 309, 312, 320, 322, 326, 329, 330, 332, 331, 333,    334, 335, 337, 338, 339, 340, 359, 360, 373, 376, 416, 419, 430,    434, 435, 437, 438, 439, and more preferably one or more of the    following modifications: T256A, H268N, E272Q, N286D, N286Q, N286S,    K290A, K290S, S298A, R301M, D312A, K320E, K320M, K320Q, K320R,    K322E, K326A, K326D, K326E, K326N, K326S, A330K, A339T, E333A,    K334A, K334E, K334H, K334L, K334M, K334Q, K334V, T335K, T335Q,    T359A, K360A, E430A; wherein such antibodies exhibit altered    effector function relative to antibodies having a wild-type Fc    region that lacks such modification;-   (IV) an amino acid modification at one or more of the following    positions: 252, 254, 265, 268, 269, 270, 278, 289, 292, 293, 294,    295, 296, 298, 300, 301, 303, 322, 324, 327, 329, 333, 335, 338,    340, 373, 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438, or    439; wherein such antibodies exhibit reduced effector function    relative to antibodies having a wild-type Fc region that lacks such    modification;-   (V) an amino acid modification at one or more of the following    positions: 280, 283, 285, 286, 290, 294, 295, 298, 300, 301, 305,    307, 309, 312, 315, 331, 333, 334, 337, 340, 360, 378, 398, or 430;    wherein such antibodies exhibit enhanced effector function relative    to antibodies having a wild-type Fc region that lacks such    modification;-   or-   (VI) an amino acid modification at one or more of the following    positions: R255A, T256A, E258A, S267A, H268A, H268N, E272A, E272Q,    N276A, D280A, E283A, H285A, N286A, N286D, N286Q, N286S, K290A,    K290S, R301M, K320E, K320M, K320Q, K320R, K322E, K326A, K326D,    K326E, K326S, A330K, P331A, T335Q, S337A, E430A; wherein such    antibodies exhibit enhanced effector function relative to antibodies    having a wild-type Fc region that lacks such modification.

In other embodiments, the invention encompasses the use of any Fcvariant known in the art, such as those disclosed in Jefferis, B. J. etal. (2002) “Interaction Sites On Human IgG-Fc For FcgammaR: CurrentModels,” Immunol. Lett. 82:57-65; Presta, L. G. et al. (2002)“Engineering Therapeutic Antibodies For Improved Function,” Biochem.Soc. Trans. 30:487-90; Idusogie, E. E. et al. (2001) “EngineeredAntibodies With Increased Activity To Recruit Complement,” J. Immunol.166:2571-75; Shields, R. L. et al. (2001) “High Resolution Mapping OfThe Binding Site On Human IgG1 For Fc Gamma RI, Fc Gamma RII, Fc GammaRill, And FcRn And Design Of IgG1 Variants With Improved Binding To TheFc gamma R,” J. Biol. Chem. 276:6591-6604; Idusogie, E. E. et al. (2000)“Mapping Of The Clq Binding Site On Rituxan, A Chimeric Antibody With AHuman IgG Fc,” J. Immunol. 164:4178-84; Reddy, M. P. et al. (2000)“Elimination Of Fc Receptor-Dependent Effector Functions Of A ModifiedIgG4 Monoclonal Antibody To Human CD4,” J. Immunol. 164:1925-1933; Xu,D. et al. (2000) “In Vitro Characterization of Five Humanized OKT3Effector Function Variant Antibodies,” Cell. Immunol. 200:16-26; Armour,K. L. et al. (1999) “Recombinant human IgG Molecules Lacking FcgammaReceptor I Binding And Monocyte Triggering Activities,” Eur. J. Immunol.29:2613-24; Jefferis, R. et al. (1996) “Modulation Of Fc(Gamma)R AndHuman Complement Activation By IgG3-Core Oligosaccharide Interactions,”Immunol. Lett. 54:101-04; Lund, J. et al. (1996) “Multiple InteractionsOf IgG With Its Core Oligosaccharide Can Modulate Recognition ByComplement And Human Fc Gamma Receptor I And Influence The Synthesis OfIts Oligosaccharide Chains,” J. Immunol. 157:4963-4969; Hutchins et al.(1995) “Improved Biodistribution, Tumor Targeting, And ReducedImmunogenicity In Mice With A Gamma 4 Variant Of Campath-1H,” Proc.Natl. Acad. Sci. (U.S.A.) 92:11980-84; Jefferis, R. et al. (1995)“Recognition Sites On Human IgG For Fc Gamma Receptors: The Role OfGlycosylation,” Immunol. Lett. 44:111-17; Lund, J. et al. (1995)“Oligosaccharide-Protein Interactions In IgG Can Modulate Recognition ByFc Gamma Receptors,” FASEB J. 9:115-19; Alegre, M. L. et al. (1994) “ANon Activating “Humanized” Anti-CD3 Monoclonal Antibody RetainsImmunosuppressive Properties In Vivo,” Transplantation 57:1537-1543;Lund et al. (1992) “Multiple Binding Sites On The CH2 Domain Of IgG ForMouse Fc Gamma R11,” Mol. Immunol. 29:53-59; Lund et al. (1991) “HumanFc Gamma RI And Fc Gamma RII Interact With Distinct But OverlappingSites On Human IgG,” J. Immunol. 147:2657-2662; Duncan, A. R. et al.(1988) “Localization Of The Binding Site For The Human High-Affinity FcReceptor On IgG,” Nature 332:563-564; U.S. Pat. Nos. 5,624,821;5,885,573; 6,194,551; 7,276,586; and 7,317,091; and PCT Publications WO00/42072 and PCT WO 99/58572.

The invention encompasses molecules comprising variant Fc regionsconsisting of or comprising any of the mutations listed in the tablebelow in Table 1.

TABLE 1 Exemplary Fc Modifications Substitutions of a Single Site S132IF241W D265N D280Q Y296T D312A L328I K334E A162V F241Y D265Q D280Y N297DW313F L328K K334H S219Y F241Y D265T G281D N297E N315I L328M K334I K222NF243D D265V G281K N297I E318K L328N K334L H224L F243H D265Y G281P N297SK320E L328P K334M T225S F243L V266A G281Y S298A K320M L328Q K334N P228EF243L V266I V282M S298D K320Q L328R K334Q P228G F243Q V266M E283A S298NK320R L328S K334V P228K F243R V266T V284E S298N K322E L328T T335K P228YF243W S267A V284L S298N V323I L328V T335Q P230A F243Y H268A V284N S298PN325A L328W I336E P230E P244H H268N V284T S298V N325D L328Y I336K P230GP245A D270E V284Y T299A N325E A330I I336Y P230Y P247G P271A H285A T299DN325F A330K S337A A231E P247L P271D N286A T299E N325G A330L A339T A231GP247V P271E N286D T299F N325H A330S M352L A231K K248M P271F N286S T299GN325I A330V T359A A231P R255A P271G K288N T299H N325K A330Y T359N A231YT256A P271H K290A T299I N325L P331A K360A P232E E258A P271I K290G T299KN325M I332A T366N P232G V262A P271K K290S T299L N325P I332D T366S P232KV262E P271L K290T T299M N325R I332E F372Y P232Y V262F P271M K290Y T299NN325S I332F F372Y E233D V262I P271N P291D T299P N325T I332G I377F E233GV262T P271Q P291E T299Q N325V I332H I377N L234I V263A P271R P291G T299RN325W I332K V379L L235D V263I P271S P291H T299S N325Y I332L V379M S239DV263M P271T P291I T299V K326A I332M K392R S239E V263T P271V P291Q T299WK326D I332N P396H S239N V264A P271W P291T T299Y K326E I332P P396L S239QV264E P271Y R292G Y300I K326E I332Q L398V V240A V264F E272A R292L Y300LK326N I332R S400P V240I V264I E272Q E294N R301M K326S I332S D401V V240MV264R V273I Q295K R301M K326T I332T S407I V240T V264T F275L Y296D V302IL328A I332V K414N F241E V264W F275W Y296E S304D L328D I332W E430A V241ID265F F275Y Y296H S304H L328E I332Y F241L D265H N276A Y296N S304L L328FE333A F241R D265I D280A Y296P S304N L328G K334A F241S D265L D280H Y296QS304T L328H K334E Substitutions of Two Sites I332E, A330L S239N/I332QV279L, P395S P396L, P217S I332E, L328D S239Q/I332D V284A, F372L P396L,P227S I332E, L328E S239Q/I332E K288N, K326N P396L, V323I I332E, L328HS239Q/I332N K288N, A330S P396L, V240A I332E, L328I S239Q/I332Q K290E,L142P P396L, L242F I332E, L328M V240I, V281M K290E, P227S P396L, P244HI332E, L328N F241L, E258G K290T, G371D P396L, T250A I332E, L328QF241L/V262I P291S, P353Q P396L, R255L I332E, L328T F243L, E318K R292P,V305I P396L, E258D I332E, L328V F243I, V379L S298A/I332E P396L, H268DI332E, N297D P243L/V264I S298N, W381R P396L, H268N I332E, N297E K246T,Y319F S298N, S407R P396L, V303I I332E, N297S K246T, P396H K317N,F423-DEL P396L, K326I S166N, K409R P247H, G285E K326E, K320E P396L,V305L P232S, S304G P247L, I377F K326E, A330T P396L, L358P S239D/I332DP247L, E389G K326E, G385E P396L, K370E S239D/I332E P247S, P396L A330V,Q419H P396L, S375C S239D/I332N P247L, L398Q K334E, E233D P396L, V379MS239D/I332Q P247L, L406F K334N, K246I P396L, N384K S239E/D265N P247L,N421K K334E, K288M P396L, K392T S239E/D265Q L251F, F372L K334E, R292LP396L, S400F S239E/I332D L251F, S415I K334E, E308D P396L, L410HS239E/I332E R255L, E318K K334E, E380D P396L, Q419H S239E/I332N R255Q,K326E K334N, P396L P396L, Q419L S239E/I332Q E258D, N384K A339V, Q347HP396L, V427A S239N/I332D V263Q, E272D K370N, S440N D399E, G402DS239N/I332E V264I/I332E T394M, V397M D399E, M428L S239N/I332N H268D,E318D P396L, K210M Substitutions of Three Sites V185M, R292L, D399EP217S, A378V, S408R K218R, G281D, G385R S192T, M252L, R301C P247L,I253N, K334N P247L, A330T, S440G V125L, V215I, S408I D312E, K327N, I378ST355N, P387S, H435Q R292L, T359N, P396L E216D, E345K, S375I P247L,A431V, S442F F275I, K334N, V348M K288N, A330S, P396L A378V, N390I, V422IF243L, R255L, E318K G316D, A378V, D399E V282E, V369I, L406F K334E,T359N, T366S N315I, V379M, T394M V397M, T411A, S415N K288N, A330S, P396LP247L, W313R, E388G T223I, T256S, L406F F243I, V379L, G420V R301H,K340E, D399E K246N, P396L, Q419R A231V, Q386H, V412M K326I, P396L, S408NP217A, T359A, P396L E216D, K334R, S375I K210M, K261N, P396L V215I,K290V, P396L T335N, P387S, H435Q A330V, G427M, K438R V263Q, E272D, Q419HK246I, Q362H, K370E K222E, V263Q, S298N N276Y, T393N, W417R K334E,E380D, G446V E233G, P247S, L306P D270E, G316D, R416G V303I, V369F, M428LS219T, T225K, D270E D270E, K392T, P396L K246E, V284M, V308A R292P,F243L, V305I R255L, D270E, P396L E293V, Q295E, A327T V284M, R292L, K370NV240A, D270E, P396L Y319F, P352L, P396L D270E, K370E, P396L 270E, P396L,Q419HD K290T, N390I, P396L P247L, D270E, N421K S239D, A330L, I332EN297D, A330Y, I332E Y296D, N297D, I332E S239D, A330Y, I332E N297D,T299L, I332E Y296E, N297D, I332E S239D, I332E, A330I N297D, T299I, I332EY296H, N297D, I332E S239D, N297D, I332E N297D, T299L, I332E Y296N,N297D, I332E S239D, S298A, I332E N297D, T299V, I332E Y296Q, N297I, I332ES239D, V2641I, I332E F243L, V262I, V264W Y296T, N297D, I332E S239E,N297D, I332E D265F, N297E, I332E P230A, E233D, I332E S239E, V264I, I332ED265Y, N297D, I332E P244H, P245A, P247V S239N, A330L, I332E V264E,N297D, I332E V264I, A330Y, I332E S239N, A330Y, I332E V264I, A330L, I332EV264I, S298A, I332E S239Q, V264I, I332E Substitutions of Four SitesA141V, H268L, K288E, P291S T256S, V305I, K334E, N390S E258D, T289A,H310Y, Y407V D280E, S354F, A431D, L441I K334E, T359N, T366S, Q386RP343S, P353L, S375I, S383N K326Q, K334E, T359N, T366S E269K, K290N,Q311R, H433Y K288R, T307A, K344E, P396L K290E, V369A, T393A, P396LV273I, K326E, L328I, P396L K210N, K222I, K320M, P396L F275L, Q362H,N384K, P396L S219T, T225K, D270E, K360R V282L, A330V, H433Y, T436RP243L, S254T, A330V, N361D R255L, D270E, Y300L, P396L, F243L, D270E,K392N, P396L R255L, D270E, R292G, P396L F243L, R255L, D270E, P396LV284M, S298N, K334E, R355W S239D, D265F, N297D, I332E D265Y, N297D,T299L, I332E S239D, D265H, N297D, I332E F241E, F2430, V262T, V264FS239D, D265I, N297D, I332E F241E, F243R, V262E, V264R S239D, 0265L,N297D, I332E F241E, F243Y, V262T, V264R S239D, D265T, N297D, I332EF241L, F243L, V262I, V264I S239D, D265V, N297D, I332E F241R, F2430,V262T, V264R S239D, D265Y, N297D, I332E F241W, F243W, V262A, V264AS239D, N297D, I332E, A330Y F241Y, F243Y, V262T, V264T S239D, N297D,I332E, K326E N297D, I332E, S239D, A330L S239D, N297D, I332E, L235DN297D, S298A, A330Y, I332E S239D, V264I, A330L, I332E S239D, A330Y,I332E, K326E S239D, V264I, S298A, I332E S239D, A330Y, I332E, K326TS239E, V264I, A330Y, I332E S239D, A330Y, I332E, L234I S239D, A330Y,I332E, V264T S239D, A330Y, I332E, L235D S239D, A330Y, I332E, V266IS239D, A330Y, I332E, V240I Substitutions of Five Sites V284M, S298N,K334E, R355W, R416T K147T, Y202M, F275I, K334N, V348M P217S, V305I,I309L, N390H, P396L T335N, K370E, A378V, T394M, S424L F243L, V305I,A378D, P396L, F404S P244H, L358M, V379M, N384K, V397M K222N, T335N,K370E, A378V, T394M P244A, K326I, C367R, S375I, K447T L235P, S304G,V305I, V323I, V382M C229Y, A287T, V379M, P396L, L443V F241E, F2430,V262T, V264E, I332E F241R, F243Q, V262T, V264R, I332E F241E, F243R,V262E, V264R, I332E S239E, V264I, S298A, A330Y, I332E F241E, F243Y,V262T, V264R, I332E Substitutions of More Than Five Sites D221E, D270E,V308A, Q311H, P396L, G402D T215P, K274N, A287G, K334N, L365V, P396LF241Y, F243Y, V262T, V264T, N297D, I332E N297D, T299F, I332E, N297D,T299H, I332E D221Y, M252I, A330G, A339T, T359N, V422I, H433L S239D,N297D, I332E, A330Y, F241S, F243H, V262T, V264T K133M, F149Y, K205E,R214I, K218E, S383N, N384K, T256N, V262L

In specific embodiments, the variant Fc region of such antiB7-H3antibodies has:

-   (1) a leucine at position 247, a lysine at position 421 and a    glutamic acid at position 270;-   (2) a threonine at position 392, a leucine at position 396, a    glutamic acid at position 270, and a leucine at position 243-   (3) a histidine at position 419, a leucine at position 396, and a    glutamic acid at position 270;-   (4) a histidine at position 419, a leucine at position 396, a    glutamic acid at position 270, and a leucine at position 243;-   (5) an alanine at position 240, a leucine at position 396, and a    glutamic acid at position 270;-   (6) a lysine at position 255 and a leucine at position 396;-   (7) a lysine at position 255, a leucine at position 396, and a    glutamic acid at position 270;-   (8) a lysine at position 255, a leucine at position 396, a glutamic    acid at position 270, and a lysine at position 300;-   (9) a lysine at position 255, a leucine at position 396, a glutamic    acid at position 270, and a glycine at position 292;-   (10) a lysine at position 255, a leucine at position 396, a glutamic    acid at position 270, and a leucine at position 243;-   (11) a glutamic acid at position 370, a leucine at position 396, and    a glutamic acid at position 270;-   (12) a glutamic acid at position 270, an aspartic acid at position    316, and a glycine at position 416;-   (13) a leucine at position 243, a proline at position 292, an    isoleucine at position 305, and a leucine at position 396;-   (14) a leucine at position 243, a glutamic acid at position 270, an    asparagine at position 392 and a leucine at position 396;-   (15) a leucine at position 243, a leucine at position 255, a    glutamic acid at position 270 and a leucine at position 396;-   (16) a glutamine at position 297;-   or-   (17) any combination of the foregoing (1)-(16) substitutions.

In some embodiments, the molecules of the invention further comprise oneor more glycosylation sites, so that one or more carbohydrate moietiesare covalently attached to the molecule. Preferably, the molecules ofthe invention with one or more glycosylation sites and/or one or moremodifications in the Fc region confer or have an enhanced antibodymediated effector function, e.g., enhanced ADCC activity, compared to aparent antibody. In some embodiments, the invention further comprisesmolecules comprising one or more modifications of amino acids that aredirectly or indirectly known to interact with a carbohydrate moiety ofthe antibody, including but not limited to amino acids at positions 241,243, 244, 245, 245, 249, 256, 258, 260, 262, 264, 265, 296, 299, and301. Amino acids that directly or indirectly interact with acarbohydrate moiety of an antibody are known in the art, see, e.g.,Jefferis et al., 1995 Immunology Letters, 44: 111-7, which isincorporated herein by reference in its entirety.

In another embodiment, the invention encompasses molecules that havebeen modified by introducing one or more glycosylation sites into one ormore sites of the molecules, preferably without altering thefunctionality of the molecules, e.g., binding activity to target antigenor FcγR. Glycosylation sites may be introduced into the variable and/orconstant region of the molecules of the invention. As used herein,“glycosylation sites” include any specific amino acid sequence in anantibody to which an oligosaccharide (i.e., carbohydrates containing twoor more simple sugars linked together) will specifically and covalentlyattach. Oligosaccharide side chains are typically linked to the backboneof an antibody via either N- or O-linkages. N-linked glycosylationrefers to the attachment of an oligosaccharide moiety to the side chainof an asparagine residue. O-linked glycosylation refers to theattachment of an oligosaccharide moiety to a hydroxyamino acid, e.g.,serine, threonine. The molecules of the invention may comprise one ormore glycosylation sites, including N-linked and O-linked glycosylationsites. Any glycosylation site for N-linked or O-linked glycosylationknown in the art may be used in accordance with the instant invention.An exemplary N-linked glycosylation site that is useful in accordancewith the methods of the present invention is the amino acid sequence:Asn-X-Thr/Ser, wherein X may be any amino acid and Thr/Ser indicates athreonine or a serine. Such a site or sites may be introduced into amolecule of the invention using methods well known in the art to whichthis invention pertains (see for example, IN VITRO MUTAGENESIS,RECOMBINANT DNA: A SHORT COURSE, J. D. Watson, et al. W.H. Freeman andCompany, New York, 1983, chapter 8, pp. 106-116, which is incorporatedherein by reference in its entirety. An exemplary method for introducinga glycosylation site into a molecule of the invention may comprise:modifying or mutating an amino acid sequence of the molecule so that thedesired Asn-X-Thr/Ser sequence is obtained.

In some embodiments, the invention encompasses methods of modifying thecarbohydrate content of a molecule of the invention by adding ordeleting a glycosylation site. Methods for modifying the carbohydratecontent of antibodies are well known in the art and encompassed withinthe invention, see, e.g., U.S. Pat. No. 6,218,149; EP 0 359 096 B1; U.S.Publication No. US 2002/0028486; WO 03/035835; U.S. Publication No.2003/0115614; U.S. Pat. No. 6,218,149; U.S. Pat. No. 6,472,511; all ofwhich are incorporated herein by reference in their entirety. In otherembodiments, the invention encompasses methods of modifying thecarbohydrate content of a molecule of the invention by deleting one ormore endogenous carbohydrate moieties of the molecule. In a specificembodiment, the invention encompasses shifting the glycosylation site ofthe Fc region of an antibody, by modifying positions adjacent to 297. Ina specific embodiment, the invention encompasses modifying position 296so that position 296 and not position 297 is glycosylated.

Effector function can also be modified by techniques such as byintroducing one or more cysteine residues into the Fc region, therebyallowing interchain disulfide bond formation in this region to occur,resulting in the generation of a homodimeric antibody that may haveimproved internalization capability and/or increased complement-mediatedcell killing and ADCC (Caron, P. C. et al. (1992) “Engineered HumanizedDimeric Forms Of IgG Are More Effective Antibodies,” J. Exp. Med.176:1191-1195; Shopes, B. (1992) “A Genetically Engineered Human IgGMutant With Enhanced Cytolytic Activity,” J. Immunol. 148(9):2918-2922.Homodimeric antibodies with enhanced anti-tumor activity may also beprepared using heterobifunctional cross-linkers as described in Wolff,E. A. et al. (1993) “Monoclonal Antibody Homodimers: Enhanced AntitumorActivity In Nude Mice,” Cancer Research 53:2560-2565. Alternatively, anantibody can be engineered which has dual Fc regions and may therebyhave enhanced complement lysis and ADCC capabilities (Stevenson, G. T.et al. (1989) “A Chimeric Antibody With Dual Fc Regions (bisFabFc)Prepared By Manipulations At The IgG Hinge,” Anti-Cancer Drug Design3:219-230).

E. B7-H3 DART (Dual Affinity Retargeting Reagents)

As discussed above, the present invention additionally encompasses“DART” (dual affinity retargeting reagent) molecules that comprise atleast two polypeptide chains which form at least two epitope bindingsites, at least one of which specifically binds to B7-H3.

In preferred embodiments, the first polypeptide chain of the DARTcomprises:

-   -   (i) a domain (A) comprising a binding region of a light chain        variable domain of a first immunoglobulin (VL1) specific for an        epitope (1);    -   (ii) a domain (B) comprising a binding region of a heavy chain        variable domain of a second immunoglobulin (VH2) specific for an        epitope (2); and    -   (iii) a domain (C).        The second polypeptide chain of such a DART comprises:    -   (i) a domain (D) comprising a binding region of a light chain        variable domain of the second immunoglobulin (VL2) specific for        epitope (2);    -   (ii) a domain (E) comprising a binding region of a heavy chain        variable domain of the first immunoglobulin (VH1) specific for        epitope (1); and    -   (iii) a domain (F).        The DART domains (A) and (B) do not associate with one another        to form an epitope binding site. Similarly, the DART domains (D)        and (E) do not associate with one another to form an epitope        binding site. Rather, DART domains (A) and (E) associate to form        a binding site that binds epitope (1); said DART domains (B)        and (D) associate to form a binding site that binds said epitope        (2). Domains (C) and (F) are covalently associated together.

Each polypeptide chain of the DART molecule comprises a VL domain and aVH domain, which are covalently linked such that the domains areconstrained from self assembly. Interaction of two of the polypeptidechains will produce two VL-VH pairings, forming two epitope bindingsites, i.e., a bivalent molecule. Neither the VH or VL domain isconstrained to any position within the polypeptide chain, i.e.,restricted to the amino (N) or carboxy (C) terminus, nor are the domainsrestricted in their relative positions to one another, i.e., the VLdomain may be N-terminal to the VH domain and vice-versa. The onlyrestriction is that a complimentary polypeptide chain be available inorder to form functional DARTs. Where the VL and VH domains are derivedfrom the same antibody, the two complimentary polypeptide chains may beidentical. For example, where the binding domains are derived from anantibody specific for epitope A (i.e., the binding domain is formed froma VL_(A)-VH_(A) interaction), each polypeptide will comprise a VH_(A)and a VL_(A). Homodimerization of two polypeptide chains of the antibodywill result in the formation two VL_(A)-VH_(A) binding sites, resultingin a bivalent monospecific antibody. Where the VL and VH domains arederived from antibodies specific for different antigens, formation of afunctional bispecific DART requires the interaction of two differentpolypeptide chains, i.e., formation of a heterodimer. For example, for abispecific DART, one polypeptide chain will comprise a VL_(A) and aVH_(B); homodimerization of said chain will result in the formation oftwo VL_(A)-VH_(B) binding sites, either of no binding or ofunpredictable binding. In contrast, where two differing polypeptidechains are free to interact, e.g., in a recombinant expression system,one comprising a VL_(A) and a VH_(B) and the other comprising a VL_(B)and a VH_(A), two differing binding sites will form: VL_(A)-VH_(A) andVL_(B)-VH_(B). For all DART polypeptide chain pairs, the possibly ofmisalignment or mis-binding of the two chains is a possibility, i.e.,interaction of VL-VL or VH-VH domains; however, purification offunctional diabodies is easily managed based on the immunospecificity ofthe properly dimerized binding site using any affinity based methodknown in the art or exemplified herein, e.g., affinity chromatography.

One or more of the polypeptide chains of the DART may optionallycomprise an Fc domain domain or portion thereof (e.g. a CH2 domain, orCH3 domain) The Fc domain or portion thereof may be derived from anyimmunoglobulin isotype or allotype including, but not limited to, IgA,IgD, IgG, IgE and IgM. In preferred embodiments, the Fc domain (orportion thereof) is derived from IgG. In specific embodiments, the IgGisotype is IgG1, IgG2, IgG3 or IgG4 or an allotype thereof. In oneembodiment, the diabody molecule comprises an Fc domain, which Fc domaincomprises a CH2 domain and CH3 domain independently selected from anyimmunoglobulin isotype (i.e. an Fc domain comprising the CH2 domainderived from IgG and the CH3 domain derived from IgE, or the CH2 domainderived from IgG1 and the CH3 domain derived from IgG2, etc.). The Fcdomain may be engineered into a polypeptide chain comprising the diabodymolecule of the invention in any position relative to other domains orportions of said polypeptide chain (e.g., the Fc domain, or portionthereof, may be c-terminal to both the VL and VH domains of thepolypeptide of the chain; may be n-terminal to both the VL and VHdomains; or may be N-terminal to one domain and c-terminal to another(i.e., between two domains of the polypeptide chain)).

The Fc domains in the polypeptide chains of the DART moleculespreferentially dimerize, resulting in the formation of a DART moleculethat exhibits immunoglobulin-like properties, e.g., Fc-FcγR,interactions. Fc comprising diabodies may be dimers, e.g., comprised oftwo polypeptide chains, each comprising a VH domain, a VL domain and anFc domain. Dimerization of said polypeptide chains results in a bivalentDART comprising an Fc domain, albeit with a structure distinct from thatof an unmodified bivalent antibody. Such DART molecules will exhibitaltered phenotypes relative to a wild-type immunoglobulin, e.g., alteredserum half-life, binding properties, etc. In other embodiments, DARTmolecules comprising Fc domains may be tetramers. Such tetramerscomprise two ‘heavier’ polypeptide chains, i.e., a polypeptide chaincomprising a VL, a VH and an Fc domain, and two ‘lighter’ polypeptidechains, i.e., polypeptide chain comprising a VL and a VH. The lighterand heavier chains interact to form a monomer, and said monomersinteract via their unpaired Fc domains to form an Ig-like molecule. Suchan Ig-like DART is tetravalent and may be monospecific, bispecific ortetraspecific.

Formation of a tetraspecific diabody molecule as described suprarequires the interaction of four differing polypeptide chains. Suchinteractions are difficult to achieve with efficiency within a singlecell recombinant production system, due to the many variants ofpotential chain mispairings. One solution to decrease the probability ofmispairings, is to engineer “knobs-into-holes” type mutations into thedesired polypeptide chain pairs. Such mutations favor heterodimerizationover homodimerization. For example, with respect to Fc-Fc-interactions,an amino acid substitution (preferably a substitution with an amino acidcomprising a bulky side group forming a ‘knob’, e.g., tryptophan) can beintroduced into the CH2 or CH3 domain such that steric interference willprevent interaction with a similarly mutated domain and will obligatethe mutated domain to pair with a domain into which a complementary, oraccommodating mutation has been engineered, i.e., ‘the hole’ (e.g., asubstitution with glycine). Such sets of mutations can be engineeredinto any pair of polypeptides comprising the diabody molecule, andfurther, engineered into any portion of the polypeptides chains of saidpair. Methods of protein engineering to favor heterodimerization overhomodimerization are well known in the art, in particular with respectto the engineering of immunoglobulin-like molecules, and are encompassedherein (see e.g., Ridgway et al. (1996) “‘Knobs-Into-Holes’ EngineeringOf Antibody CH3 Domains For Heavy Chain Heterodimerization,” ProteinEngr. 9:617-621, Atwell et al. (1997) “Stable Heterodimers FromRemodeling The Domain Interface Of A Homodimer Using A Phage DisplayLibrary,” J. Mol. Biol. 270: 26-35, and Xie et al. (2005) “A New FormatOf Bispecific Antibody: Highly Efficient Heterodimerization, ExpressionAnd Tumor Cell Lysis,” J. Immunol. Methods 296:95-101; each of which ishereby incorporated herein by reference in its entirety.

The invention also encompasses diabody molecules comprising variant Fcor variant hinge-Fc domains (or portion thereof), which variant Fcdomain comprises at least one amino acid modification (e.g.substitution, insertion deletion) relative to a comparable wild-type Fcdomain or hinge-Fc domain (or portion thereof). Molecules comprisingvariant Fc domains or hinge-Fc domains (or portion thereof) (e.g.,antibodies) normally have altered phenotypes relative to moleculescomprising wild-type Fc domains or hinge-Fc domains or portions thereof.The variant phenotype may be expressed as altered serum half-life,altered stability, altered susceptibility to cellular enzymes or alteredeffector function as assayed in an NK dependent or macrophage dependentassay. Fc domain modifications identified as altering effector functionare disclosed above.

The present invention also encompasses molecules comprising a hingedomain. The hinge domain be derived from any immunoglobulin isotype orallotype including IgA, IgD, IgG, IgE and IgM. In preferred embodiments,the hinge domain is derived from IgG, wherein the IgG isotype is IgG1,IgG2, IgG3 or IgG4, or an allotype thereof. Said hinge domain may beengineered into a polypeptide chain comprising the diabody moleculetogether with an Fc domain such that the diabody molecule comprises ahinge-Fc domain. In certain embodiments, the hinge and Fc domain areindependently selected from any immunoglobulin isotype known in the artor exemplified herein. In other embodiments the hinge and Fc domain areseparated by at least one other domain of the polypeptide chain, e.g.,the VL domain. The hinge domain, or optionally the hinge-Fc domain, maybe engineered in to a polypeptide of the invention in any positionrelative to other domains or portions of said polypeptide chain. Incertain embodiments, a polypeptide chain of the invention comprises ahinge domain, which hinge domain is at the C-terminus of the polypeptidechain, wherein said polypeptide chain does not comprise an Fc domain. Inyet other embodiments, a polypeptide chain of the invention comprises ahinge-Fc domain, which hinge-Fc domain is at the C-terminus of thepolypeptide chain. In further embodiments, a polypeptide chain of theinvention comprises a hinge-Fc domain, which hinge-Fc domain is at theN-terminus of the polypeptide chain.

Each domain of the polypeptide chain of the DART, i.e., the VL, VH andFc domain may be separated by a peptide linker. The peptide linker maybe 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in length. In certainembodiments the amino acid linker sequence is GGGSGGGG (SEQ ID NO: 52)encoded by the nucleic acid sequence ggaggcggat ccggaggcgg aggc (SEQ IDNO: 53). The polypeptide chains of the DART molecule may be engineeredto comprise at least one cysteine residue that will interact with acounterpart cysteine residue on a second polypeptide chain of the DARTto form an inter-chain disulfide bond. Such interchain disulfide bondsserve to stabilize the DART molecule, thereby improving expression andrecovery in recombinant systems, resulting in a stable and consistentformulation and improving the stability of the isolated and/or purifiedproduct in vivo. The cysteine residue may be introduced as a singleamino acid or as part of larger amino-acid sequence, e.g. a hingedomain, in any portion of the polypeptide chain. In a specificembodiment, the cysteine residue may be engineered to occur at theC-terminus of the polypeptide chain. In some embodiments, the cysteineresidue is introduced into the polypeptide chain within the amino acidsequence LGGC. In a specific embodiment, the C-terminus of thepolypeptide chains comprising the DART molecule of the inventioncomprises the amino acid sequence LGGC (SEQ ID NO: 54). In anotherembodiment, the cysteine residue is introduced into the polypeptidewithin an amino acid sequence comprising a hinge domain, e.g.EPKSCDKTHTCPP (SEQ ID NO: 55) or ESKYGPPCPS (SEQ ID NO: 56). In aspecific embodiment, the C-terminus of a polypeptide chain of the DARTmolecule of the invention comprises the amino acid sequence of an IgGhinge domain, e.g. SEQ ID NO: 55 or SEQ ID NO: 56. In anotherembodiment, the C-terminus of a polypeptide chain of a DART molecule ofthe invention comprises the amino acid sequence VEPKSC (SEQ ID NO: 57),which can be encoded by nucleotide sequence gttgagccca aatcttgt (SEQ IDNO: 58). In other embodiments, the cysteine residue in introduced intothe polypeptide chain within the amino acid sequence LGGCFNRGEC (SEQ IDNO: 59), which can be encoded by the nucleotide sequence ctgggaggctgcttcaacag gggagagtgt (SEQ ID NO: 60). In a specific embodiment, theC-terminus of a polypeptide chain comprising the DART of the inventioncomprises the amino acid sequence LGGCFNRGEC (SEQ ID NO: 59). In yetother embodiments, the cysteine residue in introduced into thepolypeptide chain within the amino acid sequence FNRGEC (SEQ ID NO: 61),which can be encoded by the nucleotide sequence ttcaacaggg gagagtgt (SEQID NO: 62). In a specific embodiment, the C-terminus of a polypeptidechain comprising the DART of the invention comprises the amino acidsequence FNRGEC (SEQ ID NO: 61).

In certain embodiments, the diabody molecule comprises at least twopolypeptide chains, each of which comprise the amino acid sequence LGGC(SEQ ID NO: 54) and are covalently linked by a disulfide bond betweenthe cysteine residues in the LGGC (SEQ ID NO: 54) sequences. In anotherspecific embodiment, the diabody molecule comprises at least twopolypeptide chains, one of which comprises the sequence FNRGEC (SEQ IDNO: 61) while the other comprises a hinge domain (containing at leastone cysteine residue), wherein said at least two polypeptide chains arecovalently linked by a disulfide bond between the cysteine residue inFNRGEC (SEQ ID NO: 61) and a cysteine residue in the hinge domain. Inparticular aspects, the cysteine residue responsible for the disulfidebond located in the hinge domain is Cys-128 (as numbered according toKabat EU; located in the hinge domain of an unmodified, intact IgG heavychain) and the counterpart cysteine residue in SEQ ID NO: 23 is Cys-214(as numbered according to Kabat EU; located at the C-terminus of anunmodified, intact IgG light chain) (Elkabetz et al. (2005) “CysteinesIn CH1 Underlie Retention Of Unassembled Ig Heavy Chains,” J. Biol.Chem. 280:14402-14412). In yet other embodiments, the at least onecysteine residue is engineered to occur at the N-terminus of the aminoacid chain. In still other embodiments, the at least one cysteineresidue is engineered to occur in the linker portion of the polypeptidechain of the diabody molecule. In further embodiments, the VH or VLdomain is engineered to comprise at least one amino acid modificationrelative to the parental VH or VL domain such that said amino acidmodification comprises a substitution of a parental amino acid withcysteine.

In still another aspect of this embodiment, the Domain (C) of the firstpolypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO:57), derived from the hinge domain of a human IgG, and which can beencoded by the nucleotide sequence gttgagccca aatcttgt (SEQ ID NO: 58).In another aspect of this embodiment, the Domain (F) of the secondpolypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO:57). In certain aspects of this embodiment, Domain (C) of the firstpolypeptide chain comprises the C-terminal 6 amino acids of the humankappa light chain, FNRGEC (SEQ ID NO: 61); and Domain (F) of the secondpolypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO:57) or a hinge domain. In other aspects of this embodiment, Domain (F)of the second polypeptide chain comprises the C-terminal 6 amino acidsof the human kappa light chain, FNRGEC (SEQ ID NO: 61); and Domain (C)of the first polypeptide chain comprises the amino acid sequence VEPKSC(SEQ ID NO: 57) or a hinge domain.

As will be appreciated in view of the foregoing, the individualpolypeptides of a bispecific DART can form two species of homodimers andone species of heterodimer. In one embodiment of the present invention,a charged polypeptide can be added to the C-terminus of one, or morepreferably, both DART polypeptides. By selecting charged polypeptides ofopposite charge for the individual polypeptides of the bispecific DART,the inclusion of such charged polypeptides favors formation ofheterodimers and lessens formation of homodimers. Preferably, apositively charged polypeptide will contain a substantial content ofarginine, glutamine, histidine and/or lysine (or mixtures of such aminoacids) and a negatively charged polypeptide will contain a substantialcontent of aspartate or glutamate (or a mixture of such amino acids).Positively charged polypeptides containing a substantial content oflysine and negatively charged polypeptides containing a substantialcontent of glutamate are particularly preferred. In order to maximizethe electrostatic attraction between such opposingly chargedpolypeptides, it is preferred to employ polypeptides capable ofspontaneously assuming a helical conformation.

Thus, in a preferred embodiment, a positively charged, “E-coil” will beappended to one of the polypeptides being used to form a bispecific DARTand a negatively charged “K-coil” will be appended to the second of theDART's polypeptides. A particularly preferred E-coil will have thesequence: (EVAALEK)₄ [i.e. (SEQ ID NO: 63)EVAALEKEVAALEKEVAALEKEVAALEK]. A particularly preferred K-coil will havethe sequence: (KVAALKE)₄ [i.e. (SEQ ID NO: 64)KVAALKEKVAALKEKVAALKEKVAALKE].

A preferred DART polypeptide possessing such an E-coil will have thegeneral sequence: [VL Domain]-[GGGSGGGG]-[VH Domain]-[(EVAALEK)₄]-GGGNS,where VL is the DART's variable light Ig domain, GGGSGGGG is SEQ ID NO:52, VH is the DART's variable heavy Ig domain, (EVAALEK)₄ is SEQ ID NO:63, and GGGNS is SEQ ID NO: 65. A preferred DART polypeptide possessingsuch a K-coil will have the general sequence: [VL Domain]-[GGGSGGGG]-[VHDomain]-[(KVAALKE)₄]-GGGNS, where VL is the DART's variable light Igdomain, GGGSGGGG is SEQ ID NO: 52, VH is the DART's variable heavy Igdomain, (KVAALKE)₄ is SEQ ID NO: 64, and GGGNS is SEQ ID NO: 65.

In a further embodiment, Fc-regions can be linked to the E and/or Kcoils of E-coil or K-coil DARTs. Furthering the separation between theFc regions and the DART VH domain of an Fc-containing DART is desirablein cases in which a less separated arrangement of such domains resultsin diminished interaction between such domains and their binding ligandsor otherwise interferes with DART assembly. Although separators of anyamino acid sequence may be employed, it is preferable to employseparators that form an a helix coils, so as to maximally extend andproject the Fc domain away from the variable domains. Because theabove-described coiled polypeptides of opposing charge additionallyfunction to promote heterodimer formation, such molecules areparticularly preferred separators. Such coil-containing Fc-DARTmolecules provide benefits similar to those of Fc-DARTS, includingimproved serum half-life and effector function recruitment. Theabove-described E-coil and K-coil polypeptides are particularlypreferred for this purpose. Thus, in a preferred embodiment, the E-coilFc-containing DART will have the general sequence: [VLDomain]-[GGGSGGGG]-[VH Domain]-[(EVAALEK)₄]-GGG-Fc domain starting withD234 (Kabat numbering), where VL is the DART's variable light Ig domain,GGGSGGGG is SEQ ID NO: 52, VH is the DART's variable heavy Ig domain and(EVAALEK)₄ is SEQ ID NO: 63. Similarly, in a preferred embodiment, theK-coil Fc-containing DART will have the general sequence: [VLDomain]-[GGGSGGGG]-[VH Domain]-[(KVAALKE)₄]-GGG-Fc domain starting withD234 (Kabat numbering), where VL is the DART's variable light Ig domain,GGGSGGGG is SEQ ID NO: 51, VH is the DART's variable heavy Ig domain and(KVAALKE)₄ is SEQ ID NO: 64.

As indicated above, a coil-containing DART molecule or a coil-containingFc-containing DART molecule may contain only a single such coilseparator, or it may contain more than one such separators (e.g., twoseparators, preferably of opposite charge, of which one is linked toeach of the VH domain of the DART's polypeptides). By linking the Fcregion to such separator molecule(s), the ability to make bivalent,tetravalent, etc. versions of the Fc-DART molecules by chain swapping isenhanced. Fc-DART molecules can thus be produced that form monomers ordimers depending upon whether the Fc domain is linked to one or both ofthe DART VH domains

1. Versatility of B7-H3 DART Molecules

The bispecific DARTs of the invention can simultaneously bind twoseparate and distinct epitopes. In certain embodiments the epitopes arefrom the same antigen. In other embodiments, the epitopes are fromdifferent antigens. In preferred embodiments, at least one epitopebinding site is specific for a determinant expressed on an immuneeffector cell (e.g. CD3, CD16, CD32, CD64, T-cell receptor, etc.) whichare expressed on T lymphocytes, natural killer (NK) cells or othermononuclear cells. In one embodiment, the DART molecule binds to theeffector cell determinant and also activates said effector cell. In thisregard, DART molecules of the invention may exhibit Ig-likefunctionality independent of whether they further comprise an Fc domain(e.g., as assayed in any effector function assay known in the art orexemplified herein (e.g., ADCC assay). In certain embodiments thebispecific DART of the invention binds both a cancer antigen on a tumorcell and an effector cell determinant while activating said cell. Inalternative embodiments, the bispecific DART or DART molecule of theinvention may inhibit activation of a target, e.g., effector, cell bysimultaneously binding, and thus linking, an activating and inhibitoryreceptor on the same cell (e.g., bind both CD32A and CD32B, BCR andCD32B, or IgERI and CD32B) as described supra (see, Background Section).In a further aspect of this embodiment, the bispecific DART may exhibitantiviral properties by simultaneously binding two neutralizing epitopeson a virus (e.g., RSV epitopes; WNV epitopes such as E16 and E53).

2. Universal B7-H3 DART Molecules

In one embodiment, the bispecific DART molecules of the invention may beconstructed to comprise one epitope binding domain that specificallybinds to B7-H3 and a second epitope binding domain that specificallybinds a hapten, e.g. fluorescein isothiocyanate (also known asfluoroisothiocyanate or FITC). Such a DART serves as a universal adaptor(“UDART”), able to co-ligate B7-H3 with molecules that interact withfluorescein-conjugated binding partners. For example, the FITC-reactivearm of the DART may be used to bind to an FITC labeled antibody that isbound to a non-B7-H3 target involved in intercellular clustering,intercellular recruitment, cell-free recruitment, multiple targets, etc.The chimeric mouse Fv/human Fc version of the anti-fluorescein MAb, 4420may be employed as a source of FITC-specific CDR domains (Gruber, M. etal. (1994) “Efficient Tumor Cell Lysis Mediated By A Bispecific SingleChain Antibody Expressed In Escherichia coli,” J. Immunol. 152(11):5368-5374).

3. Cell-Target Specific B7-H3 DART Molecules

The bispecific DART molecules of the invention offer uniqueopportunities to target specific cell types. For example, the bispecificDART or DART molecule can be engineered to comprise a combination ofepitope binding sites that recognize a set of antigens unique to atarget cell or tissue type. Additionally, where either or both of theindividual antigens is/are fairly common separately in other tissueand/or cell types, low affinity biding domains can be used to constructthe DART or DART molecule. Such low affinity binding domains will beunable to bind to the individual epitope or antigen with sufficientavidity for therapeutic purposes. However, where both epitopes orantigens are present on a single target cell or tissue, the avidity ofthe DART or DART molecule for the cell or tissue, relative to a cell ortissue expressing only one of the antigens, will be increased such thatsaid cell or tissue can be effectively targeted by the invention. Such abispecific molecule can exhibit enhanced binding to one or both of itstarget antigens on cells expressing both of said antigens relative to amonospecific DART or an antibody with a specificity to only one of theantigens.

For Example, the B7-H3 specific DARTS of the present invention may beconstructed to comprise a domain that is a binding ligand for theNatural Killer Group 2D (NKG2D) receptor. The NKG2D receptor isexpressed on all human (and other mammalian) Natural Killer cells(Bauer, S. et al. (1999) “Activation Of NK Cells And T Cells By NKG2D, AReceptor For Stress Inducible MICA,” Science 285(5428):727-729;Jamieson, A. M. et al. (2002) “The Role Of The NKG2D Immunoreceptor InImmune Cell Activation And Natural Killing,” Immunity 17(1):19-29) aswell as on all CD8⁺ T cells (Groh, V. et al. (2001) “Costimulation OfCD8a/3 T Cells By NKG2D Via Engagement By MIC Induced On Virus-InfectedCells,” Nat. Immunol. 2(3):255-260; Jamieson, A. M. et al. (2002) “TheRole Of The NKG2D Immunoreceptor In Immune Cell Activation And NaturalKilling,” Immunity 17(1):19-29). Such binding ligands, and particularlythose which are not expressed on normal cells, include thehistocompatibility 60 (H60) molecule, the product of the retinoic acidearly inducible gene-1 (RAE-1), and the murine UL16-binding protein liketranscript 1 (MULTI) (Raulet D. H. (2003) “Roles Of The NKG2DImmunoreceptor And Its Ligands,” Nature Rev. Immunol. 3:781-790;Coudert, J. D. et al. (2005) “Altered NKG2D Function In NK Cells InducedBy Chronic Exposure To Altered NKG2D Ligand-Expressing Tumor Cells,”Blood 106:1711-1717). Additional ligands reactive with human NKG2Dinclude the polymorphic MHC class I chain-related molecules MICA andMICB (Diefenbach, A. et al. (1999) “Natural Killer Cells: Stress Out,Turn On, Tune In,” Curr. Biol. 9(22):R851-R8533; Bauer, S. et al. (1999)“Activation Of NK Cells And T Cells By NKG2D, A Receptor ForStress-Inducible MICA,” Science 285(5428):727-729; Stephens, H. A.(2001) “MICA And MICB Genes: Can The Enigma Of Their Polymorphism BeResolved?” Trends Immunol. 22:378-385.

The sequence of MICA is SEQ ID NO: 66:MGLGPVFLLL AGIFPFAPPG AAAEPHSLRY NLTVLSWDGSVQSGFLTEVH LDGQPFLRCD RQKCRAKPQG QWAEDVLGNKTWDRETRDLT GNGKDLRMTL AHIKDQKEGL HSLQEIRVCEIHEDNSTRSS QHFYYDGELF LSQNLETKEW TMPQSSRAQTLAMNVRNFLK EDAMKTKTHY HAMHADCLQE LRRYLKSGVVLRRTVPPMVN VTRSEASEGN ITVTCRASGF YPWNITLSWRQDGVSLSHDT QQWGDVLPDG NGTYQTWVAT RICQGEEQRFTCYMEHSGNH STHPVPSGKV LVLQSHWQTF HVSAVAAAAIFVIIIFYVRC CKKKTSAAEG PELVSLQVLD QHPVGTSDHR DATQLGFQPL MSDLGSTGST EGAThe sequence of MICB is SEQ ID NO: 67:PHSLRYNLMV LSQDGSVQSG FLAEGHLDGQ PFLRYDRQKRRAKPQGQWAE DVLGAKTWDT ETEDLTENGQ DLRRTLTHIKDQKGGLHSLQ EIRVCEIHED SSTRGSRHFY YDGELFLSQNLETQESTVPQ SSRAQTLAMN VTNFWKEDAM KTKTHYRAMQADCLQKLQLP PMVNVICSEV SEGNITVTCR ASSFYPRNITLTWRQDGVSL SHNTQQWGDV LPDGNGTYQT WVATRIRQGEEQRFTCYMEH SGNHGTHPVP SGKALVLQSQ RTDFPYVSAA MPCFVIIIIL CVPCCKKKTS AAEGP

Alternatively, the DART molecules of the present invention may beconstructed to comprise a domain that is a binding ligand for the T-cellreceptor (“TCR”) or for CD3 (the T-cell co-receptor). The TCR isnatively expressed by CD4+ or CD8+ T-cells, and permits such cells torecognize antigenic peptides that are bound and presented by class I orclass II MHC proteins of antigen-presenting cells. Recognition of a pMHC(peptide-MHC) complex by a TCR initiates the propagation of a cellularimmune response that leads to the production of cytokines and the lysisof the antigen-presenting cell (see, e.g., Armstrong, K. M. et al.(2008) “Conformational Changes And Flexibility In T-Cell ReceptorRecognition Of Peptide MHC Complexes,” Biochem. J. 415(Pt 2):183-196;Willemsen, R. (2008) “Selection Of Human Antibody Fragments DirectedAgainst Tumor T-Cell Epitopes For Adoptive T-Cell Therapy,” Cytometry A.73(11):1093-1099; Beier, K. C. et al. (2007) “Master Switches Of T-CellActivation And Differentiation,” Eur. Respir. J. 29:804-812; Mallone, R.et al. (2005) “Targeting T Lymphocytes For Immune Monitoring AndIntervention In Autoimmune Diabetes,” Am. J. Ther. 12(6):534-550). CD3is the receptor that binds to the TCR (Thomas, S. et al. (2010)“Molecular Immunology Lessons From Therapeutic T-Cell Receptor GeneTransfer,” Immunology 129(2):170-177; Guy, C. S. et al. (2009)“Organization Of Proximal Signal Initiation At The TCR:CD3 Complex,”Immunol. Rev. 232(1):7-21; St. Clair, E. W. (Epub 2009 Oct. 12) “NovelTargeted Therapies For Autoimmunity,” Curr. Opin. Immunol.21(6):648-657; Baeuerle, P. A. et al. (Epub 2009 Jun. 9) “BispecificT-Cell Engaging Antibodies For Cancer Therapy,” Cancer Res.69(12):4941-4944; Smith-Garvin, J. E. et al. (2009) “T Cell Activation,”Annu. Rev. Immunol. 27:591-619; Renders, L. et al. (2003) “EngineeredCD3 Antibodies For Immunosuppression,” Clin. Exp. Immunol.133(3):307-309).

By constructing such DART molecules to additionally comprise at leastone epitope-binding domain capable of binding to, for example, areceptor present on the surface of a target cell, such DART moleculeswill thus be capable of binding to the target cells and thereby causethe target cells to display the binding ligand for the Natural KillerGroup 2D (NKG2D) receptor or to the TCR (whichever is present on thetarget cell-bound DART) (see, e.g., Germain, C. et al. (2008)“Redirecting NK Cells Mediated Tumor Cell Lysis By A New RecombinantBifunctional Protein,” Prot. Engineer. Design Selection 21(11):665-672).Such DARTs can be used to redirect any desired target cell into a cellthat is a target of NK cell-mediated cell lysis or T-cell mediatedcytotoxicity. In one embodiment, the epitope-binding domain of the DARTcapable of binding to a receptor present on the surface of a target cellis an epitope that binds to a tumor-associated antigen so as to redirectsuch cancer cells into substrates for NK cell-mediated cell lysis orT-cell mediated cytotoxicity. Of particular interest is atumor-associated antigens that is a breast cancer antigen, an ovariancancer antigen, a prostate cancer antigen, a cervical cancer antigen, apancreatic carcinoma antigen, a lung cancer antigen, a bladder cancerantigen, a colon cancer antigen, a testicular cancer antigen, aglioblastoma cancer antigen, an antigen associated with a B cellmalignancy, an antigen associated with multiple myeloma, an antigenassociated with non-Hodgkins lymphoma, or an antigen associated withchronic lymphocytic leukemia.

Suitable tumor-associated antigens for such use include A33 (acolorectal carcinoma antigen; Almqvist, Y. 2006, Nucl Med Biol.November; 33(8):991-998); B1 (Egloff, A. M. et al. 2006, Cancer Res.66(1):6-9); BAGE (Bodey, B. 2002 Expert Opin Biol Ther. 2(6):577-84);beta-catenin (Prange W. et al. 2003 J Pathol. 201(2):250-9); CA125(Bast, R. C. Jr. et al. 2005 Int J Gynecol Cancer 15 Suppl 3:274-81);CD5 (Calin, G. A. et al. 2006 Semin Oncol. 33(2):167-73; CD19(Troussard, X. et al. 1998 Hematol Cell Ther. 40(4):139-48); CD20(Thomas, D. A. et al. 2006 Hematol Oncol Clin North Am. 20(5):1125-36);CD22 (Kreitman, R. J. 2006 AAPS J. 18; 8(3):E532-51); CD23 (Rosati, S.et al. 2005 Curr Top Microbiol Immunol. 5; 294:91-107); CD25 (Troussard,X. et al. 1998 Hematol Cell Ther. 40(4):139-48); CD27 (Bataille, R. 2006Haematologica 91(9):1234-40); CD28 (Bataille, R. 2006 Haematologica91(9):1234-40); CD36 (Ge, Y. 2005 Lab Hematol. 11(1):31-7); CD40/CD154(Messmer, D. et al. 2005 Ann N Y Acad Sci. 1062:51-60); CD45 (Jurcic, J.G. 2005 Curr Oncol Rep. 7(5):339-46); CD56 (Bataille, R. 2006Haematologica 91(9):1234-40); CD79a/CD79b (Troussard, X. et al. 1998Hematol Cell Ther. 40(4):139-48; Chu, P. G. et al. 2001 ApplImmunohistochem Mol Morphol. 9(2):97-106); CD103 (Troussard, X. et al.1998 Hematol Cell Ther. 40(4):139-48); CDK4 (Lee, Y. M. et al. 2006 CellCycle 5(18):2110-4); CEA (carcinoembryonic antigen; Mathelin, C. 2006Gynecol Obstet Fertil. 34(7-8):638-46; Tellez-Avila, F. I. et al. 2005Rev Invest Clin. 57(6):814-9); CTLA4 (Peggs, K. S. et al. 2006 Curr OpinImmunol. 18(2):206-13); EGF-R (epidermal growth factor receptor; Adenis,A. et al. 2003 Bull Cancer. 90 Spec No:S228-32); Erb (ErbB1; ErbB3;ErbB4; Zhou, H. et al. 2002 Oncogene 21(57):8732-40; Rimon, E. et al.2004 Int J Oncol. 24(5):1325-38); GAGE (GAGE-1; GAGE-2; Akcakanat, A. etal. 2006 Int J Cancer. 118(1):123-8); GD2/GD3/GM2 (Livingston, P. O. etal. 2005 Cancer Immunol Immunother. 54(10):1018-25); gp100 (Lotem, M. etal. 2006 J Immunother. 29(6):616-27); HER-2/neu (Kumar, Pal S et al.2006 Semin Oncol. 33(4):386-91); human papillomavirus-E6/humanpapillomavirus-E7 (DiMaio, D. et al. 2006 Adv Virus Res. 66:125-59; KSA(17-1A) (Ragupathi, G. 2005 Cancer Treat Res. 123:157-80); MAGE (MAGE-1;MAGE-3; (Bodey, B. 2002 Expert Opin Biol Ther. 2(6):577-84); MART(Kounalakis, N. et al. 2005 Curr Oncol Rep. 7(5):377-82; MUC-1(Mathelin, C. 2006 Gynecol Obstet Fertil. 34(7-8):638-46); MUM-1(Castelli, C. et al. 2000 J Cell Physiol. 182(3):323-31);N-acetylglucosaminyltransferase (Dennis, J. W. 1999 Biochim BiophysActa. 6; 1473(1):21-34); p15 (Gil, J. et al. 2006 Nat Rev Mol Cell Biol.7(9):667-77); PSA (prostate specific antigen; Cracco, C. M. et al. 2005Minerva Urol Nefrol. 57(4):301-11); PSMA (Ragupathi, G. 2005 CancerTreat Res. 123:157-80); sTn (Holmberg, L. A. 2001 Expert Opin Biol Ther.1(5):881-91); TNF-receptor (TNF-α receptor, TNF-β receptor; or TNF-γreceptor; van Horssen, R. et al. 2006 Oncologist. 11(4):397-408;Gardnerova, M. et al. 2000 Curr Drug Targets. 1(4):327-64); or VEGFreceptor (O'Dwyer. P. J. 2006 Oncologist. 11(9):992-8).

Additional tumor-associated antigens for such use (and publicationsdisclosing specifically reactive antibodies for such antigens) includeADAM-9 (United States Patent Publication No. 2006/0172350; PCTPublication No. WO 06/084075); ALCAM (PCT Publication No. WO 03/093443);Carboxypeptidase M (United States Patent Publication No. 2006/0166291);CD46 (U.S. Pat. No. 7,148,038; PCT Publication No. WO 03/032814);Cytokeratin 8 (PCT Publication No. WO 03/024191); Ephrin receptors (andin particular EphA2 (U.S. Pat. No. 7,569,672; PCT Publication No. WO06/084226); Integrin Alpha-V-Beta-6 (PCT Publication No. WO 03/087340);JAM-3 (PCT Publication No. WO 06/084078); KID3 (PCT Publication No. WO05/028498); KID31 (PCT Publication No. WO 06/076584); LUCA-2 (UnitedStates Patent Publication No. 2006/0172349; PCT Publication No. WO06/083852); Oncostatin M (Oncostatin Receptor Beta) (U.S. Pat. No.7,572,896; PCT Publication No. WO 06/084092); PIPA (U.S. Pat. No.7,405,061; PCT Publication No. WO 04/043239); ROR1 (U.S. Pat. No.5,843,749); and the Transferrin Receptor (U.S. Pat. No. 7,572,895; PCTPublication No. WO 05/121179).

Also of interest are antigens specific to particular infectious agents,e.g., viral agents including, but not limited to human immunodeficiencyvirus (HIV), hepatitis B virus (HBV), influenza, human papilloma virus(HPV), foot and mouth (coxsackieviruses), the rabies virus, herpessimplex virus (HSV), and the causative agents of gastroenteritis,including rotaviruses, adenoviruses, caliciviruses, astroviruses andNorwalk virus; bacterial agents including, but not limited to E. coli,Salmonella thyphimurium, Pseudomonas aeruginosa, Vibrio cholerae,Neisseria gonorrhoeae, Helicobacter pylori, Hemophilus influenzae,Shigella dysenteriae, Staphylococcus aureus, Mycobacterium tuberculosisand Streptococcus pneumoniae, fungal agents and parasites such asGiardi.

In some embodiments, molecules of the invention are engineered tocomprise an altered glycosylation pattern or an altered glycoformrelative to the comparable portion of the template molecule. Engineeredglycoforms may be useful for a variety of purposes, including, but notlimited to, enhancing effector function. Engineered glycoforms may begenerated by any method known to one skilled in the art, for example byusing engineered or variant expression strains, by co-expression withone or more enzymes, for example, DI N-acetylglucosaminyltransferase III(GnTI11), by expressing a DART of the invention in various organisms orcell lines from various organisms, or by modifying carbohydrate(s) afterthe DART has been expressed and purified. Methods for generatingengineered glycoforms are known in the art, and include but are notlimited to those described in Umana et al. (1999) “Engineered GlycoformsOf An Antineuroblastoma IgG1 With Optimized Antibody-Dependent CellularCytotoxic Activity,” Nat. Biotechnol 17:176-180; Davies et al. (2001)“Expression Of GnTIII In A Recombinant Anti-CD20 CHO Production CellLine: Expression Of Antibodies With Altered Glycoforms Leads To AnIncrease In Adcc Through Higher Affinity For Fc Gamma RIII,” BiotechnolBioeng 74:288-294; Shields et al. (2002) “Lack Of Fucose On Human IgG1N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII AndAntibody-Dependent Cellular Toxicity,” J Biol Chem 277:26733-26740;Shinkawa et al. (2003) “The Absence Of Fucose But Not The Presence OfGalactose Or Bisecting N-Acetylglucosamine Of Human IgG1 Complex-TypeOligosaccharides Shows The Critical Role Of Enhancing Antibody-DependentCellular Cytotoxicity,” J Biol Chem 278:3466-3473) U.S. Pat. No.6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT WO00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO 02/30954A1;Potillegent™ technology (Biowa, Inc. Princeton, N.J.); GlycoMAb™glycosylation engineering technology (GLYCART biotechnology AG, Zurich,Switzerland); each of which is incorporated herein by reference in itsentirety. See, e.g., WO 00061739; EA01229125; US 20030115614; Okazaki etal. (2004) “Fucose Depletion From Human IgG1 Oligosaccharide EnhancesBinding Enthalpy And Association Rate Between IgG1 And FcGammaRIIIA,”JMB, 336: 1239-49 each of which is incorporated herein by reference inits entirety.

The invention further encompasses incorporation of unnatural amino acidsto generate the DARTs of the invention. Such methods are known to thoseskilled in the art such as those using the natural biosyntheticmachinery to allow incorporation of unnatural amino acids into proteins,see, e.g., Wang et al. (2002) “Expanding The Genetic Code,” Chem. Comm1: 1-11; Wang et al. (2001) “Expanding The Genetic Code Of Escherichiacoli,” Science, 292: 498-500; van Hest et al. (2001) “Protein-BasedMaterials, Toward A New Level Of Structural Control,” Chem. Comm 19:1897-1904, each of which is incorporated herein by reference in itsentirety. Alternative strategies focus on the enzymes responsible forthe biosynthesis of amino acyl-tRNA, see, e.g., Tang et al. (2001)“Biosynthesis Of A Highly Stable Coiled-Coil Protein ContainingHexafluoroleucine In An Engineered Bacterial Host,” J. Am. Chem. Soc.123(44): 11089-11090; Kiick et al. (2001) “Identification Of An ExpandedSet Of Translationally Active Methionine Analogues In Escherichia coli,”FEBS Lett. 502(1-2):25-30; each of which is incorporated herein byreference in its entirety. In some embodiments, the inventionencompasses methods of modifying a VL, VH or Fc domain of a molecule ofthe invention by adding or deleting a glycosylation site. Methods formodifying the carbohydrate of proteins are well known in the art andencompassed within the invention, see, e.g., U.S. Pat. No. 6,218,149; EP0 359 096 B1; U.S. Publication No. US 2002/0028486; WO 03/035835; U.S.Publication No. 2003/0115614; U.S. Pat. No. 6,218,149; U.S. Pat. No.6,472,511; all of which are incorporated herein by reference in theirentirety.

VIII. Methods of Using B7-H3 Modulators and Anti-B7-H3 Antibodies forTherapeutic Purposes

Monoclonal antibodies to B7-H3 may be used for therapeutic purposes inindividuals with cancer or other diseases. Therapy with anti-B7-H3antibodies can involve formation of complexes both in vitro and in vivoas described above. In one embodiment, monoclonal antibody anti-B7-H3can bind to and reduce the proliferation of cancerous cells. It isunderstood that the antibody is administered at a concentration thatpromotes binding at physiological (e.g., in vivo) conditions. In anotherembodiment, monoclonal antibodies to B7-H3 can be used for immunotherapydirected at cancerous cells of different tissues such as colon, lung,breast, prostate, ovary, pancreas, kidney and other types of cancer suchas sarcoma. In another embodiment, monoclonal antibody anti-B7-H3 alonecan bind to and reduce cell division in the cancer cell. In anotherembodiment, monoclonal antibody anti-B7-H3 can bind to cancerous cellsand delay the development of metastasis. In yet another embodiment, anindividual with cancer is given palliative treatment with anti-B7-H3antibody. Palliative treatment of a cancer individual involves treatingor lessening the adverse symptoms of the disease, or iatrogenic symptomsresulting from other treatments given for the disease without directlyaffecting the cancer progression. This includes treatments for easing ofpain, nutritional support, sexual problems, psychological distress,depression, fatigue, psychiatric disorders, nausea, vomiting, etc.

In such situations, the anti-B7-H3 antibody may be administered withagents that enhance or direct an individual's own immune response, suchas an agent that strengthens ADCC.

In yet another embodiment, anti-B7-H3 antibody be conjugated to orassociated with a radioactive molecule, toxin (e.g., calicheamicin),chemotherapeutic molecule, liposomes or other vesicles containingchemotherapeutic compounds and administered to an individual in need ofsuch treatment to target these compounds to the cancer cell containingthe antigen recognized by the antibody and thus eliminate cancerous ordiseased cells. Without being limited to any particular theory, theanti-B7-H3 antibody is internalized by the cell bearing B7-H3 at itssurface, thus delivering the conjugated moiety to the cell to induce thetherapeutic effect. In yet another embodiment, the antibody can beemployed as adjuvant therapy at the time of the surgical removal of acancer expressing the antigen in order to delay the development ofmetastasis. The antibody can also be administered before surgery(neoadjuvant therapy) in an individual with a tumor expressing theantigen in order to decrease the size of the tumor and thus enable orsimplify surgery, spare tissue during surgery, and/or decrease theresulting disfigurement.

Cell cycle dosing is contemplated in the practice of this invention. Insuch embodiments, a chemotherapeutic agent is used to synchronize thecell cycle of the tumor or other target diseased cells at apre-determined stage. Subsequently, administration of the anti-B7-H3antibody of this invention (alone or with an additional therapeuticmoiety) is made. In alternative embodiments, an anti-B7-H3 antibody isused to synchronize the cell cycle and reduce cell division prior toadministration of a second round of treatment; the second round may beadministration of an anti-B7-H3 antibody and/or an additionaltherapeutic moiety.

Chemotherapeutic agents include radioactive molecules, toxins, alsoreferred to as cytotoxins or cytotoxic agents, which includes any agentthat is detrimental to the viability of cancerous cells, agents, andliposomes or other vesicles containing chemotherapeutic compounds.Examples of suitable chemotherapeutic agents include but are not limitedto 1-dehydrotestosterone, 5-fluorouracil decarbazine, 6-mercaptopurine,6-thioguanine, actinomycin D, adriamycin, aldesleukin, alkylatingagents, allopurinol sodium, altretamine, amifostine, anastrozole,anthramycin (AMC)), anti-mitotic agents, cis-dichlorodiamine platinum(II) (DDP) cisplatin), diaminodichloroplatinum, anthracyclines,antibiotics, antimetabolites, asparaginase, BCG live (intravesical),betamethasone sodium phosphate and betamethasone acetate, bicalutamide,bleomycin sulfate, busulfan, calcium leucouorin, calicheamicin,capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU),Chlorambucil, Cisplatin, Cladribine, Colchicin, conjugated estrogens,Cyclophosphamide, Cyclothosphamide, Cytarabine, Cytarabine, cytochalasinB, Cytoxan, Dacarbazine, Dactinomycin, dactinomycin (formerlyactinomycin), daunirubicin HCL, daunorucbicin citrate, denileukindiftitox, Dexrazoxane, Dibromomannitol, dihydroxyanthracindione,Docetaxel, dolasetronmesylate, doxorubicin HCL, dronabinol, E. coliL-asparaginase, emetine, epoetin alpha, Erwinia L-asparaginase,esterified estrogens, estradiol, estramustine phosphate sodium, ethidiumbromide, ethinyl estradiol, etidronate, etoposide citrororum factor,etoposide phosphate, filgrastim, floxuridine, fluconazole, fludarabinephosphate, fluorouracil, flutamide, folinic acid, gemcitabine HCL,glucocorticoids, goserelin acetate, gramicidin D, granisetron HCL,hydroxyurea, idarubicin HCL, ifosfamide, interferon alpha-2b, irinotecanHCL, letrozole, leucovorin calcium, leuprolide acetate, levamisole HCL,lidocaine, lomustine, maytansinoid, mechlorethamine HCL,medroxyprogesterone acetate, megestrol acetate, melphalan HCL,mercaptipurine, mesna, methotrexate, methyltestosterone, mithramycin,mitomycin C, mitotane, mitoxantrone, nilutamide, octreotide acetate,ondansetron HCL, paclitaxel, pamidronate disodium, pentostatin,pilocarpine HCL, plimycin, polifeprosan 20 (with carmustine implant),porfimer sodium, procaine, procarbazine HCL, propranolol, rituximab,sargramostim, streptozotocin, tamoxifen, taxol, teniposide, tenoposide,testolactone, tetracaine, thioepa chlorambucil, thioguanine, thiotepa,topotecan HCL, toremifene citrate, trastuzumab, tretinoin, valrubicin,vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate.

In a preferred embodiment, the cytotoxin is especially effective individing or rapidly dividing cells, such that non-dividing cells arerelatively spared from the toxic effects.

The antibodies of the invention can be internalized within the diseasedor carcinoma cells to which they bind and are therefore particularlyuseful for therapeutic applications, for example, delivering into thecells toxins that need to be internalized for their adverse activity.Examples of such toxins include, but are not limited to, saporin,calicheamicin, auristatin, and maytansinoid.

The antibodies or polypeptides of the invention can be associated(including conjugated or linked) to a radioactive molecule, a toxin, orother therapeutic agents, or to liposomes or other vesicles containingtherapeutic agents covalently or non-covalently, directly or indirectly.The antibody may be linked to the radioactive molecule, the toxin, orthe chemotherapeutic molecule at any location along the antibody so longas the antibody is able to bind its target B7-H3.

A toxin or a chemotherapeutic agent may be administered concurrentlywith (before, after, or during administration), or coupled (e.g.,covalently bonded) to a suitable monoclonal antibody either directly orindirectly (e.g., via a linker group, or, alternatively, via a linkingmolecule with appropriate attachment sites, such as a platform moleculeas described in U.S. Pat. No. 5,552,391). The toxin and chemotherapeuticagent of the present invention can be coupled directly to the particulartargeting proteins using methods known in the art. For example, a directreaction between an agent and an antibody is possible when eachpossesses a substituent capable of reacting with the other. For example,a nucleophilic group, such as an amino or sulfhydryl group, on one maybe capable of reacting with a carbonyl-containing group, such as ananhydride or an acid halide, or with an alkyl group containing a goodleaving group (e.g., a halide) on the other.

The antibodies or polypeptides can also be linked to a chemotherapeuticagent via a microcarrier. The term “microcarrier” refers to abiodegradable or a non-biodegradable particle which is insoluble inwater and which has a size of less than about 150 μm, 120 μm or 100 μmin size, more commonly less than about 50-60 μm, preferably less thanabout 10, 5, 2.5, 2 or 1.5 μm. Microcarriers include “nanocarriers”,which are microcarriers have a size of less than about 1 μm, preferablyless than about 500 nm. Such particles are known in the art. Solid phasemicrocarriers may be particles formed from biocompatible naturallyoccurring polymers, synthetic polymers or synthetic copolymers, whichmay include or exclude microcarriers formed from agarose or cross-linkedagarose, as well as other biodegradable materials known in the art.Biodegradable solid phase microcarriers may be formed from polymerswhich are degradable (e.g., poly(lactic acid), poly(glycolic acid) andcopolymers thereof) or erodible (e.g., poly(orthoesters), such as3,9-diethylidene-2,4,8,10-tetraoxaspiro [5.5]undecane (DETOSU) orpoly(anhydrides), such as poly(anhydrides) of sebacic acid) undermammalian physiological conditions. Microcarriers may also be liquidphase (e.g., oil or lipid based), such as liposomes, iscoms(immune-stimulating complexes, which are stable complexes ofcholesterol, and phospholipid, adjuvant-active saponin) without antigen,or droplets or micelles found in oil-in-water or water-in-oil emulsions,provided the liquid phase microcarriers are biodegradable. Biodegradableliquid phase microcarriers typically incorporate a biodegradable oil, anumber of which are known in the art, including squalene and vegetableoils. Microcarriers are typically spherical in shape, but microcarriersthat deviate from spherical shape are also acceptable (e.g., ellipsoid,rod-shaped, etc.). Due to their insoluble nature (with respect towater), microcarriers are filterable from water and water-based(aqueous) solutions.

The antibody or polypeptide conjugates of the present invention mayinclude a bifunctional linker that contains both a group capable ofcoupling to a toxic agent or chemotherapeutic agent and a group capableof coupling to the antibody. A linker can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker can be cleavable or non-cleavable. Alinker can also serve to increase the chemical reactivity of asubstituent on an agent or an antibody, and thus increase the couplingefficiency. An increase in chemical reactivity may also facilitate theuse of agents, or functional groups on agents, which otherwise would notbe possible. The bifunctional linker can be coupled to the antibody bymeans that are known in the art. For example, a linker containing anactive ester moiety, such as an N-hydroxysuccinimide ester, can be usedfor coupling to lysine residues in the antibody via an amide linkage. Inanother example, a linker containing a nucleophilic amine or hydrazineresidue can be coupled to aldehyde groups produced by glycolyticoxidation of antibody carbohydrate residues. In addition to these directmethods of coupling, the linker can be indirectly coupled to theantibody by means of an intermediate carrier such as an aminodextran. Inthese embodiments the modified linkage is via either lysine,carbohydrate, or an intermediate carrier. In one embodiment, the linkeris coupled site-selectively to free thiol residues in the protein.Moieties that are suitable for selective coupling to thiol groups onproteins are well known in the art. Examples include disulfidecompounds, α-halocarbonyl and α-halocarboxyl compounds, and maleimides.When a nucleophilic amine function is present in the same molecule as an&#945;-halo carbonyl or carboxyl group the potential exists forcyclization to occur via intramolecular alkylation of the amine Methodsto prevent this problem are well known to one of ordinary skill in theart, for example by preparation of molecules in which the amine and&#945;-halo functions are separated by inflexible groups, such as arylgroups or trans-alkenes, that make the undesired cyclizationstereochemically disfavored. See, for example, U.S. Pat. No. 6,441,163for preparation of conjugates of maytansinoids and antibody via adisulfide moiety.

One of the cleavable linkers that can be used for the preparation ofantibody-drug conjugates is an acid-labile linker based on cis-aconiticacid that takes advantage of the acidic environment of differentintracellular compartments such as the endosomes encountered duringreceptor mediated endocytosis and the lysosomes. See, for example, Shen,W. C. et al. (1981) (“cis-Aconityl Spacer Between Daunomycin AndMacromolecular Carriers: A Model Of pH-Sensitive Linkage Releasing DrugFrom A Lysosomotropic Conjugate,” Biochem. Biophys. Res. Comtnun.102:1048-1054 (1981)) for the preparation of conjugates of daunorubicinwith macromolecular carriers; Yang et al. (1988) (“Pharmacokinetics AndMechanism Of Action Of A Doxorubicin Monoclonal Antibody 9.2.27Conjugate Directed To A Human Melanoma Proteoglycan,” J. Natl. Canc.Inst. 80:1154-1159) for the preparation of conjugates of daunorubicin toan anti-melanoma antibody; Dillman et al. (1988) (“Superiority Of AnAcid-Labile Daunorubicin Monoclonal Antibody Immunoconjugate Compared ToFree Drug,” Cancer Res. 48:6097-6102) for using an acid-labile linker ina similar fashion to prepare conjugates of daunorubicin with an anti-Tcell antibody; and Trouet et al. (1982) “A Covalent Linkage BetweenDaunorubicin And Proteins That Is Stable In Serum And Reversible ByLysosomal Hydrolases, As Required For A Lysosomotropic Drug-CarrierConjugate: In Vitro And In Vivo Studies,” Proc. Natl. Acad. Sci.(U.S.A.) 79:626-629) for linking daunorubicin to an antibody via apeptide spacer arm.

An antibody (or polypeptide) of this invention may be conjugated(linked) to a radioactive molecule or toxin by any method known to theart. For a discussion of methods for radiolabeling antibody (see, CANCERTHERAPY WITH MONOCLONAL ANTIBODIES, D. M. Goldenberg (Ed.) CRC Press,Boca Raton, 1995). Suitable toxins include taxanes, maytansinoids,auristatins (e.g., monomethyl auristatin (MMAE), monomethyl auristatin F(MMAF), auristatin E (AE), etc.) (such as those disclosed in U.S. Pat.Nos. 5,208,020; 5,416,064; 6,333,410; 6,340,701; 6,372,738; 6,436,931;6,441,163; 6,596,757; 7,276,497; 7,585,857; or 7,851,432),calicheamicin, anthracyclines (e.g., doxorubicin), CC-1065 analog,docetaxel; cathepsin B or E; ricin, gelonin, Pseudomonas exotoxin,diphtheria toxin, and RNase; tiuxetan or toxic radioisotope (such as⁹⁰Y; ¹³¹I, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, ²¹²Bi, ²¹³Bi, ²²⁵Ac, etc.).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described in U.S. Pat. No.4,676,980. The formation of cross-linked antibodies can target theimmune system to specific types of cells, for example, cancer ordiseased cells expressing B7-H3.

This invention also provides methods of delaying development ofmetastasis in an individual with cancer (including, but not limited to,prostate, lung, or kidney cancer) using an anti-B7-H3 antibody or otherembodiments that bind to B7-H3 in combination with a chemotherapeuticagent, or linked to a chemotherapeutic agent. In some embodiments, theantibody is a humanized or chimeric form of a non-human anti-B7-H3antibody.

In yet another embodiment, the antibody can be employed as adjuvanttherapy at the time of the surgical removal of a cancer expressing theantigen in order to delay the development of metastasis. The antibody orantibody associated with a chemotherapeutic agent can also beadministered before surgery (neoadjuvant therapy) in an individual witha tumor expressing the antigen in order to decrease the size of thetumor and thus enable or simplify surgery, spare tissue during surgery,and/or decrease the resulting disfigurement.

In yet another embodiment, any of the B7-H3 binding compositionsdescribed herein can bind to B7-H3-expressing cancerous cells and inducean active immune response against the cancerous cells expressing B7-H3.In some cases, the active immune response can cause the death of thecancerous cells (e.g., antibody binding to cancer cells inducingapoptotic cell death), or inhibit the growth (e.g., block cells cycleprogression) of the cancerous cells. In other cases, any of the novelantibodies described herein can bind to cancerous cells and antibodydependent cellular cytotoxicity (ADCC) can eliminate cancerous cells towhich anti-B7-H3 binds. Accordingly, the invention provides methods ofstimulating an immune response comprising administering any of thecompositions described herein.

In some cases, antibody binding can also activate both cellular andhumoral immune responses and recruit more natural killer cells orincreased production of cytokines (e.g., IL-2, IFN-gamma, IL-12,TNF-alpha, TNF-beta, etc.) that further activate an individual's immunesystem to destroy cancerous cells. In yet another embodiment, anti-B7-H3antibodies can bind to cancerous cells, and macrophages or otherphagocytic cell can opsonize the cancerous cells.

Various formulations of anti-B7-H3 antibodies may be used foradministration. In some embodiments, anti-B7-H3 antibodies may beadministered neat. In addition to the pharmacologically active agent,the compositions of the present invention may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries that are well known in the art and are relatively inertsubstances that facilitate administration of a pharmacologicallyeffective substance or which facilitate processing of the activecompounds into preparations that can be used pharmaceutically fordelivery to the site of action. For example, an excipient can give formor consistency, or act as a diluent. Suitable excipients include but arenot limited to stabilizing agents, wetting and emulsifying agents, saltsfor varying osmolarity, encapsulating agents, buffers, and skinpenetration enhancers.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts. In addition, suspensions of the active compounds asappropriate for oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substances thatincrease the viscosity of the suspension and include, for example,sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally,the suspension may also contain stabilizers. Liposomes can also be usedto encapsulate the agent for delivery into the cell.

The pharmaceutical formulation for systemic administration according tothe invention may be formulated for enteral, parenteral or topicaladministration. Indeed, all three types of formulation may be usedsimultaneously to achieve systemic administration of the activeingredient. Excipients as well as formulations for parenteral andnonparenteral drug delivery are set forth in REMINGTON: THE SCIENCE ANDPRACTICE OF PHARMACY, 21st Edition, Lippincott Williams & WilkinsPublishing (2005). Suitable formulations for oral administration includehard or soft gelatin capsules, pills, tablets, including coated tablets,elixirs, suspensions, syrups or inhalations and controlled release formsthereof. Generally, these agents are formulated for administration byinjection {e.g., intraperitoneally, intravenously, subcutaneously,intramuscularly, etc.), although other forms of administration (e.g.,oral, mucosal, etc) can be also used. Accordingly, anti-B7-H3 antibodiesare preferably combined with pharmaceutically acceptable vehicles suchas saline, Ringer's solution, dextrose solution, and the like.

The particular dosage regimen, i.e., dose, timing and repetition, willdepend on the particular individual and that individual's medicalhistory. Generally, a dose of at least about 100 μg/kg body weight, morepreferably at least about 250 μg/kg body weight, even more preferably atleast about 750 μg/kg body weight, even more preferably at least about 3mg/kg body weight, even more preferably at least about 5 mg/kg bodyweight, even more preferably at least about 10 mg/kg body weight isadministered.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. Antibodies, which arecompatible with the human immune system, such as humanized antibodies orfully human antibodies, may be used to prolong half-life of the antibodyand to prevent the antibody being attacked by the host's immune system.Frequency of administration may be determined and adjusted over thecourse of therapy, and is based on reducing the number of cancerouscells, maintaining the reduction of cancerous cells, reducing theproliferation of cancerous cells, or delaying the development ofmetastasis. Alternatively, sustained continuous release formulations ofanti-B7-H3 antibodies may be appropriate. Various formulations anddevices for achieving sustained release are known in the art.

In one embodiment, dosages for anti-B7-H3 antibodies may be determinedempirically in individuals who have been given one or moreadministration(s). Individuals are given incremental dosages of ananti-B7-H3 antibody. To assess efficacy of anti-B7-H3 antibodies, amarker of the specific cancer disease state can be followed. Theseinclude direct measurements of tumor size via palpation or visualobservation, indirect measurement of tumor size by x-ray or otherimaging techniques; an improvement as assessed by direct tumor biopsyand microscopic examination of the tumor sample; the measurement of anindirect tumor marker (e.g., PSA for prostate cancer), a decrease inpain or paralysis; improved speech, vision, breathing or otherdisability associated with the tumor; increased appetite; or an increasein quality of life as measured by accepted tests or prolongation ofsurvival. It will be apparent to one of skill in the art that the dosagewill vary depending on the individual, the type of cancer, the stage ofcancer, whether the cancer has begun to metastasize to other location inthe individual, and the past and concurrent treatments being used.

Other formulations include suitable delivery forms known in the artincluding, but not limited to, carriers such as liposomes. See, forexample, Mahato et al. (1997) “Cationic Lipid-Based Gene DeliverySystems: Pharmaceutical Perspectives,” Pharm. Res. 14:853-859. Liposomalpreparations include, but are not limited to, cytofectins, multilamellarvesicles and unilamellar vesicles.

In some embodiments, more than one antibody may be present. Theantibodies can be monoclonal or polyclonal. Such compositions maycontain at least one, at least two, at least three, at least four, atleast five different antibodies that are reactive against carcinomas,adenocarcinomas, sarcomas, or adenosarcomas. Anti-B7-H3 antibody can beadmixed with one or more antibodies reactive against carcinomas,adenocarcinomas, sarcomas, or adenosarcomas in organs including but notlimited to ovary, breast, lung, prostate, colon, kidney, skin, thyroid,bone, upper digestive tract, and pancreas. In one embodiment, a mixtureof different anti-B7-H3 antibodies is used. A mixture of antibodies, asthey are often denoted in the art, may be particularly useful intreating a broader range of population of individuals.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the present invention unless specified.

Example 1 Immunohistocompatability Investigations

A panel of 49 mAbs was generated from tumor cell/fetal progenitor cellimmunizations. The antibodies were evaluated for their ability toexhibit differential IHC staining of tumor tissue relative to normal,non-cancerous tissue, capability of use in primate (and particularlycynomolgus monkey) models of antibody efficacy, levels of affinity andantigen specificity and levels of immunomodulatory activity and cellularinternalization. 21 of the mAbs were initially identified by MS analysisand/or binding to B7-H3-CHO cells. The remaining 28 mAbs were identifiedby rescreening the library by ELISA with B7-H3 protein. Characteristicsof 46 of the 49 members of the panel are provided in Table 2.

TABLE 2 ATCC BIACORE ™ Cyno B7-H3 Name Isotype IHC Array InternalizationU-DART Analysis Binding BRCA84D IgG1/k 2a 2 + + + ++ TDH6 IgG1/k 2a1 + + +/− + TES7 IgG1/k 2a 1 + + + − BRCA68D IgG1/k 2b 3 + + ++ ++BRCA69D IgG1/k 2b 3 + + ++ ++ GB8 IgG1/k 2b 3 + + + ++ SG27 IgG2b/k 2b1 + + + OVCA22 IgG1/k 2c 3 + + +/− + PRCA157 IgG1/k 2c 2 + + ++ BLABIgG1/k 2c +/− + ++ ++ KID35 IgG1/k 2c 2 ++ LUCA50 IgG2a/k 2c 1 + ++OVCA21 IgG1/k 2c 1 + + + PRCA135 IgG1/k 2c 3 + ++ SG24 IgG2a/k 2c 3 ++++ TDH5 IgG1/k 2c 3 + ++ ++ BCCA66 IgG1/k 2c 2 + − RECA13 IgG1/k 2c 3 +− RECA9 IgG1/k 2c 3 + − PRCA123 IgG1/k 2c/3 3 + ++ BRCA126 IgG1/k 3/FBRCA192 IgG1/k 3/F BRCA34 IgG1/k 3/F KID1 IgG1/k 3/F ND + + + KID13IgG2a/k 3/F 3 ++ LU14 IgG2b/k 3/F − LUCA1 IgG1/k 3/F 1 + + ++ ++ MCLY42IgG2a/k 3/F ++ MCLY46 IgG1/k 3/F ++ OVCA40 IgG1/k 3/F ++ PA20 IgG1/k3/F + ++ − PA40 IgG2b/k 3/F 3 − PA41 IgG1/k 3/F 3 PRO6 IgG1/k 3/F 2 −RECA22 IgG1/k 3/F 3 − + SAL3 IgG2a/k 3/F +++ ++ SG20 IgG1/k 3/F + SG29IgG1/k 3/F ++ SKIN2 IgG1/k 3/F 3 +++ ++ STO5 IgG2b/k 3/F 3 ++ + TDH36IgG1/k 3/F 2 ++ TDH37 IgG1/k 3/F 3 + TDH4 IgG1/k 3/F +++ ++ ++ TDH40IgG2b/k 3/F 3 ++ TDH44 IgG2b/k 3/F ++ OVCA25 IgG1/k 3/F 3 +

IHC staining confirmed that the panel comprised antibodies that eliciteda strong tumor to normal tissue binding differential in many of theidentified antibodies, exhibited a range of binding properties byBIACORE™ analysis, exhibited reactivity to range of overlapping andnon-overlapping epitopes and exhibited a range of specificity to 4Ig vs.2Ig B7-H3. The characteristics of the nine best candidates are shown inTable 3 and Table 4.

TABLE 3 Colon Lung Prostate Breast Name Normal Tissue Cancer CancerCancer Cancer BRCA84D Colon 1+ 1231 * 1130 112 1111 Lung 1+ Liver 1+TDH6 Colon 1+ 1110 * 1010 111 1011 Panc 1+ Kidney 1+ Lung 1+ Liver 1+TES7   1.5     1.75  3   3 BRCA68D Panc 1+ 2321 * 3332 333 3333 Kidney1+ Lung 1+ Liver 2+ BRCA69D Colon 1+ 2231 * 3231 333 3333 Panc 1+ Kidney1+ Liver 1+ GB8 SG27 Colon 1+ 1221 * 1120 222 1122 Panc 1+ Kidney 1+Liver 1+ OVCA22 Colon 2+ 1122     3131 ** 222 3233 Panc 2+ Liver 2+PRCA157 Colon 2+ 2231 * 3231 333 2333 Liver 2+ Skin 2+ * +str also; **str 3+

TABLE 4 2Ig/4Ig Epitope Name Specificity Group BRCA84D 4Ig/2Ig 1 TDH64Ig/2Ig 2 TES7 4Ig 3 BRCA68D 4Ig/2Ig 4 BRCA69D 4Ig/2Ig 4 GB8 4Ig/2Ig 5SG27 4Ig/2Ig 6 OVCA22 4Ig 7 PRCA157 4Ig/2Ig 8

Table 5 provides a summary of the activity profiles of these antibodies.

TABLE 5 Normal Tumor Cyno Tissue Tumor/Normal Tissue Cross- BindingUDART Name Staining Differential Positive Reactivity IHC* BIACORE ™Activity BRCA84D 1 1 Tumor Positive 78 ++ ++ Stroma (not 1:1) bv TES7 11 Tumor negative 1250 + ++ Stroma bv BRCA68D 3 3 Tumor Positive 20 +++++ (1:1) BRCA69D 3 3 Tumor Positive 20 +++ ++ Stroma (1:1) GB8 2/3 3/4Tumor ND, 625 + + (Adrenal Stroma +recomb. ND) SG27 2/3 ND ND ND,20000 + + +recomb. OVCA22 1 1 Tumor Negative + 2500 + ++ Stroma recomb.PRCA157 2 3 Tumor Positive 20 ND ++ Stroma (1:1) bv *OptimalConcentration in ng/ml; ND, Not Determined

An analysis of the activities of the antibodies shown in Table 6revealed that their respective profiles differed and that each antibodywas associated with both advantages and disadvantages relative to eachother (Table 6).

TABLE 6 Antibody Advantages Disadvantages BRCA84D #1 normal tissuestaining Cyno cross-reactivity #1 tumor/normal differential not 1:1stain tumor, stroma, BV mid affinity, unique binding site (titratablebinding) BRCA68D #3 normal tissue staining Stain tumor only #3tumor/normal differential Cyno cross-reactivity 1:1 high affinity PotentUDART activity BRCA69D #3 normal tissue staining #3 tumor/normaldifferential Cyno cross-reactivity 1:1 stain tumor, stroma high affinityPotent UDART activity PRCA157 #2 normal tissue staining BIACORE ™ #3tumor/normal differential Cyno cross-reactivity 1:1 stain tumor, stroma,BV Potent UDART activity TES7 #1/2 normal tissue staining No Cynocross-reactivity #1/2 tumor/normal differential low affinity staintumor, stroma, BV 4Ig specific Potent UDART activity OVCA22 #1/2 normaltissue staining No Cyno cross-reactivity #1/2 tumor/normal differentiallow affinity low affinity stain tumor, stroma 4Ig specific Potent UDARTactivity GB8 #2/3 normal tissue staining Cyno cross-reactivity not(adrenal not determined) determined low affinity #3/4 tumor/normaldifferential stain tumor, stroma Modest UDART activity SG27 #2/3 normaltissue staining Cyno cross-reactivity not tumor/normal differential notdetermined low affinity determinedModest UDART activity

Because BRCA84D, BRCA68D, BRCA69D and PRCA157 exhibited cleaner normaltissue IHC profiles, stronger tumor/normal IHC differential, moderate tostrong binding (BIACORE™/IHC), cross-reactivity to B7-H3 of cynomolgusmonkeys, and potent UDART activity, these antibody species were selectedfor further development. These antibodies differed from TEST and OVCA2,which exhibited low affinity (in the BIACORE™ assay), and nocross-reactivity to B7-H3 of cynomolgus monkeys. These antibodiesdiffered from SG27, which exhibited low affinity (in the BIACORE™assay), poor IHC performance (weak binding) and lower UDART activity.These antibodies differed from GB8, which exhibited low affinity (in theBIACORE™ assay), poor tumor/normal IHC differential, and lower UDARTactivity.

Using Caki-2 and Hs700T positive control cells, IHC investigationsrevealed that each of the antibodies exhibited a different optimalconcentration and a different differential concentration relative to oneanother (Table 7).

TABLE 7 Optimal IHC Differential IHC Antibody ConcentrationConcentration BRCA84D 0.625 μg/ml 0.078 μg/ml BRCA68D 0.156 μg/ml 0.0195μg/ml BRCA69D 0.156 μg/ml 0.0195 μg/ml PRCA157 0.078 μg/ml 0.0195 μg/mlTES7 5 μg/ml 1.25 μg/ml OVCA22 10 μg/ml 2.5 μg/ml * GB8 1.25 μg/ml 0.625μg/ml SG27 20 μg/ml Not Determined **  TDH6 20 μg/ml Not Determined*** * OVCA22 only showed binding to caki2 cells, did not show binding toHs700T cells. Optimization decision based on the binding of caki2 cells.** Because SG27 did not show consistent titration analysis resultsbetween two operators, low affinity and differential concentration werenot determined. *** TDH6 study not done due to too low affinity topositive control cells

Using the optimal and differential concentrations indicated in Table 7,the IHC responses of the B7-H3 antibodies in human tissues weredetermined. The results of these analyses for Adrenal, Liver,Pancreatic, Kidney Lung and Colon are shown in Tables 8A-8B and Tables9A-9B (all antibodies exhibited negative IHC responses for hearttissue).

TABLE 8A B7H3 mAb IHC at Optimal Concentration in Human Tissues mAbAdrenal Liver Pancreas BRCA84D Negative Sinousoid lining cells++Epithelium+ 5% 0.625 μg/ml Hepatocytes+, 5-10% Fibre++ BRCA68D Cortex+++Sinousoid lining cells++ Epithelium+ 0.156 μg/ml Hepatocytes++ (m)Fibre++ BRCA69D Cortex+++ Epithelium+ 0.156 μg/ml Hepatocytes++ (m)Fibre++ TES7 Cortex+ Sinousoid lining cells+ Epithelium+ 5% 5 μg/mlFibre++ OVCA22 Cortex+ Sinousoid lining cells++ Epithelium+ 5% 10 μg/mlHepatocytes+ (m) Fibre++ PRCA157 Cortex++ Sinousoid lining cells++Epithelium+ 5% 0.078 μg/ml Hepatocytes+ (m) Fibre++ GB8 Not DeterminedSinousoid lining cells++ Epithelium+ 1.25 μg/ml Hepatocytes+ (m) Fibre++

TABLE 8B B7H3 mAb IHC at Optimal Concentration in Human Tissues mAbKidney Lung Colon BRCA84D Negative Epithelium+ Epithelium+ 0.625 μg/ml(5-10%) BRCA68D Fibroblast+, rare Epithelium+ Mucosa++ 0.156 μg/mlBRCA69D Fibroblast+, rare Epithelium+ Mucosa+ 0.156 μg/ml TES7 NegativeNegative Epithelium+ 5 μg/ml OVCA22 Fibroblast+ Negative Epithelium+ 10μg/ml PRCA157 Negative Negative Mucosa+ 0.078 μg/ml GB8 NegativeEpithelium+ Mucosa+ 1.25 μg/ml

TABLE 9A B7H3 mAb IHC at Differential Concentration in Human Tissues mAbAdrenal Liver Pancreas BRCA84D Negative Sinousoid lining cells+ Fibre+(rare) 0.078 μg/ml BRCA68D Cortex++ Hepatocytes+ (m) Fibre+ 0.0195 μg/mlBRCA69D Cortex++ Hepatocytes+ (m) Fibre+ 0.0195 μg/ml TES7 Fibroblast+Sinousoid lining cells+ Epithelium+, 5% 1.25 μg/ml Fibre++ OVCA22Fibroblast+ Sinousoid lining cells+ Fibre+ 2.5 μg/ml PRCA157 NotDetermined Sinousoid lining cells+ Fibre+ 0.0195 μg/ml Hepatocytes+ (m)GB8 Not Determined Sinousoid lining cells++ Fibre+ 0.625 μg/mlHepatocytes+ (m)

TABLE 9B B7H3 mAb IHC at Differential Concentration in Human Tissues mAbKidney Lung Colon BRCA84D Negative Negative Epithelium+ 0.078 μg/mlBRCA68D Negative Fibrin+ (rare) Mucosa+ 0.0195 μg/ml BRCA69D NegativeNegative Mucosa+ 0.0195 μg/ml TES7 Negative Negative Epithelium+ 1.25μg/ml OVCA22 Negative Negative Epithelium+ 2.5 μg/ml PRCA157 NegativeNegative Mucosa+ 0.0195 μg/ml GB8 Negative Negative Mucosa+ 0.625 μg/ml

IHC investigations conducted using cancer specimens showed that theB7-H3 antibodies of the present invention could be used to identify anddiagnose cancer in multiple tissue sources (Table 10). In Table 10, thenumbers indicate the number of plus signs (1=+, 2=++, 3=+++); eachnumber referring to a different tested sample.

TABLE 10 Prostate Breast Lung Colon mAbs μg/ml Cancer Cancer CancerCancer BRCA84D  0.625 μg/ml 2, 2, 1 3, 3, 3, 3 2, 3, 2(stroma),2(stroma), 3, 3, 1(b.v.) 3(stroma)  0.078 μg/ml 0, 2, 3, 2 1(stroma), 1,1, 0, 1 2, 1(stroma), 2(stroma), 1(stroma), 2, 3 1(stroma), 2(stroma)BRCA68D  0.156 μg/ml 2, 3, 3, 3 2, 3, 3, 3, 3 3, 3, 2, 2 3, 3, 3, 30.0195 μg/ml 0, 1, 1, 1 0, 0, 2, 2, 1 0, 1, 1, 0 1, 1, 1, 1 BRCA69D 0.156 μg/ml 3, 3, 3 3, 3, 3, 3, 3 3, 3, 2, 3, 3, 3, 3 2(stroma) 0, 1,2, 1 1, 2, 1, 1 1, 1, 0, 1 1(b.v.), 2(stroma), 1, 1 GB8  1.25 μg/ml 2,3, 1 2, 2, 1, 2 3, 3, 0, 0 2(stroma), 2, 2, 2  0.625 μg/ml 0, 1, 1 0, 0,0, 0, 1 1, 0, 0, 0 1(stroma), 1(stroma), 0, 0 TES7    5 μg/ml 2, 3, 2, 31(stroma), 3, 3, 2, 3, 3, 2, 2 3, 3, 2 1(stroma), 1(b.v.)  1.25 μg/ml 1,2, 2, 3 1(stroma), 2, 3, 1, 3, 3, 3, 2 1(stroma), 2(stroma), 1(b.v.)2(stroma), 2 OVCA22    10 μg/ml 3, 2, 2, 1 1(stroma), 2, 3, 2,1(stroma), 2, 3, 3 1(stroma), 1, 1, 0 2(stroma)   2.5 μg/ml 1, 1, 3, 11(stroma), 2, 1, 0, 0 1, 0, 1(stroma), 2(stroma), 1(stroma), 3, 0 2, 2PRCA157  0.078 μg/ml 2, 2, 2, 3 1, 2, 2, 3, 3 2, 2, 3, 3, 1(stroma),2(stroma), 1(b.v.) 2(stroma) 0.0195 μg/ml 0, 1, 2, 1 1(stroma), 0, 0, 1,0, 0 1, 1, 2, 1(stroma), 1(stroma) 1(stroma)

For prostate, breast, colon and lung cancer cells treated with B7-H3antibody BRCA84D, tumor sample staining was present in tumor cells andstromal cells, including the tumor vasculature. In some tumor samples,stromal staining was much stronger than tumor cells. When BRCA84D mAbwas titrated to lower concentration, some cases showed reduced stainingin tumor cells, but still maintained strong stromal staining Uponstaining with BRCA84D at 0.625 μg/ml, prostate cancer cells exhibited anIHC of 3/3+; breast cancer cells exhibited an IHC of 4/4+; colon cancercells exhibited an IHC of 4/4+ and lung cancer cells exhibited an IHC of4/4+. Upon staining with BRCA84D at 0.078 μg/ml, prostate cancer cellsexhibited an IHC of 3/4+; breast cancer cells exhibited an IHC of 5/5+;colon cancer cells exhibited an IHC of 4/4+ and lung cancer cellsexhibited an IHC of 3/4+.

Normal liver was treated with B7-H3 antibody BRCA68D, and staining wasseen in hepatocytes and sinusoid lining cells. Normal pancreas stainedwith B7-H3 antibody BRCA68D exhibited multi-focal staining in collagenfibre and epithelium. Normal adrenal cells treated with B7-H3 antibodyBRCA68D, exhibited staining in the cortex. Upon staining with BRCA68D at0.156 μg/ml, gastric, renal and ovarian cancer cells all exhibited anIHC of 5/5+.

Additional IHC staining analyses were conducted on samples of gastric,kidney and ovarian cancer tissues. The results of such analyses areshown in Table 12. In Table 11, the numbers indicate the number of plussigns (1=+, 2=++, 3=+++); each number referring to a different testedsample.

TABLE 11 Gastric Kidney Ovarian mAbs μg/ml Cancer Cancer Cancer BRCA84D 0.625 μg/ml 2, 1, 2, 2, 2 1, 2, 1, 1, 1 0, 3, 1, 2, 2  0.078 μg/ml 1,0, 0, 1, 0 0, 1, 0, 1, 0, 1 0, 2, 0, 1, 1 BRCA68D  0.156 μg/ml 3, 2, 3,3, 3 3, 3, 2, 3, 3, 3 2, 3, 3, 2, 2 0.0195 μg/ml 2, 1, 2, 1, 1 2, 2, 2,2, 2, 2 1, 2, 2, 1, 1 OVCA22    10 μg/ml 3, 1, 3, 1, 1 3, 1, 2, 3, 0, 22, 3, 2, 1, 1   2.5 μg/ml 2, 0, 2, 1, 0 2, 1, 1, 2, 0, 1 1, 2, 1, 0, 1TES7    5 μg/ml 2, 1, 3, 2, 1 2, 3, 1, 2, 2, 1 1, 3, 1, 2, 2  1.25 μg/ml2, 0, 2, 1, 1 2, 2, 1, 1, 1, 1 1, 3, 1, 2, 2

In summary, all of the tested mAbs showed various degrees of stainingintensity in normal liver, pancreas, colon and lung. FIG. 1A shows theresults of IHC investigations conducted using normal pancreas, liver,lung and colon tissue specimens with BRCA84D at 0.625 μg/ml and 0.078μg/ml. Liver staining was relatively restricted in sinusoid lining cells(fibroblast and kupffer cells) with BRCA84D and TES7. OVCA22 showedmembrane hepatocyte staining besides that of the sinusoid ling cells atthe optimal concentration. However, the staining in hepatocytesdisappeared at the differential concentration. All of the other mAbsshowed staining in hepatocytes including either membrane or cytoplasmstaining at both the optimal and differential concentration. Pancreasstaining was observed in collagen fiber mainly and a small percentage ofthe epithelium (acinar cells or/and intercalated duct cells). Thestaining in epithelium diminished or disappeared at differentialconcentration. Colon staining was relatively restricted in apicalmembrane of crypt epithelium and fibroblast in mucosa. No binding wasobserved in lymphoid nodules of colon. Lung showed very weak and patchystaining in epithelium with BRCA84D, BRCA68D, BRCA69D and GB8. However,the staining disappeared at the differential concentration. No stainingwas observed in lung with TES7, OVCA22 and PRCA157 at bothconcentrations. Adrenal cortex staining was observed with almost all ofthe mAbs at optimal concentration, except BRCA84D. The staining inadrenal obviously diminished with TES7 and OVCA22 at differentialconcentration. Heart and kidney did not show obvious staining with allmAbs (FIG. 1B). In light of these properties, BRCA84D was considered thebest of the mAbs, followed in order by (2) TES7, (3) OVCA22, (4) thegroup BRCA68D, BRCA69D and PRCA157, and lastly (5) GB8.

All of the mAbs included in the study showed positive staining in 4cancer types at the optimal concentration. At the differentialconcentration, BRCA84D still maintained good staining in prostatecancer, breast cancer, and colon cancer. TES7 maintained good stainingin 4 study cancer types. The remaining mAbs showed various stainingintensities in different tumor types. Tumor sample staining was observedin tumor cells and stromal cells, including vasculature. Some tumorsamples showed positive staining only in vasculature, i.e. BRCA84D,BRCA69D, TES7, and PRCA157. Some tumors showed stronger stromal stainingthan tumor cell staining. When mAbs were titrated to lower concentrationon these samples, some cases showed diminished or no staining in tumorcells, but still maintained strong stromal staining. In general, interms of expression in human normal tissues and differential expressionin normal vs. tumor tissues, the mAb order from the best IHC performanceto the poorest performance is as follows: (1) BRCA84D, (2) TES7, (3)OVCA22, (4) the group BRCA68D, BRCA69D and PRCA157, and lastly (5) GB8.Table 12 and FIG. 2 show results for antibody BRCA84D.

TABLE 12 Cancer BRCA84D BRCA84D Tissue Type 0.625 μg/ml 0.078 μg/mlProstate 3/3+ 3/4+ Breast 4/4+ 5/5+ Colon 4/4+ 4/4+ Lung 4/4+ 3/4+

Example 2 Cynomolgus Monkey B7-H3 Cross-Reactivity

The sequence of cynomolgus monkey B7-H3 shares approximately 90%homology to its human counterpart, suggesting that the cynomolgus monkeyis an excellent model for human B7-H3 interactions. Investigations wereconducted to evaluate the cross-reactivity of B7H3 candidates BRCA84D,BRCA68D, BRCA69D, TES7, OVCA22 and PRCA157 with adrenal, liver, kidney,pancreas and lung as well as one case full term placenta of cynomolgusmonkey, in order to compare any cross-reactivity with the stainingintensity and staining patterns observed for human tissues.

Staining concentration for each tested Mab is the optimal concentrationwhich was determined in Caki-2 and Hs700T positive control cells (see,Table 8). Commercial goat anti human B7-H3 (cross-reacted with cyno) wasselected as a positive control antibody to stain cynomolgus placentaltissue. Corresponding isotype controls were applied in each run of theexperiments. The results of the investigations are shown in Table 13.

TABLE 13 mAb Adrenal (2) Liver (2) Pancreas (2) Kidney (2) Lung (2)Placenta (1) BRCA84D Negative Negative Negative Negative 1/2 Decidualcells 0.625 μg/ml Epithelium 2+ 1+ Mesenchymal cells negative BRCA68DCortex 3+ 2/2 1/2 Fibroblast Negative Decidual cells, 0.156 μg/mlHepatocytes Fiber 2+ 1+ villi, 1+ (m) Epithelium Mesenchymal Sinousoid1+ cells 3+ lining cells 1+ BRCA69D Cortex 2+ 1/2 1/2 FibroblastNegative Decidual cells 0.156 μg/ml Hepatocytes Epithelium 1+ 2+, villi,1+ (m) 1+ rare Mesenchymal cells 2+ TES7 Negative Negative NegativeNegative Negative Negative 5 μg/ml OVCA22 Negative Negative NegativeNegative negative Negative 10 μg/ml PRCA157 Cortex 1+ 1/2 NegativeNegative Negative Decidual cells 0.078 μg/ml Hepatocytes 2+, villi, 1+(m) Mesenchymal cells 1+ Note: BRCA84D showed negative staining in liverand pancreas at up to 5 μg/ml. Although OVCA22 did not bind to cynotissue in IHC, modest binding was observed to recombinant cyno B7H3 onCHO cells. IHC score in normal tissues is negative, 1+, 2+ and 3+ 4grade system; m = membrane; 2/2 = 2 of 2 cases, 1/2 = 1 of 2 cases

The investigation of BRCA84D (0.625 μg/ml) IHC staining in cynomolgusplacenta exhibited staining in decidual cells, but not in villi. Nostaining was observed in cyno liver and pancreas, however, staining ofsinousoid lining cells was observed in human liver and localized fibreand epithellium staining was observed in human pancreas tissue.

The investigation of BRCA68D (0.156 μg/ml) IHC staining in cynomolgusplacenta exhibited staining in decidual cells, mesenchymal cells(endothelium and fibroblasts) and villi. Staining was present inmembrane of hepatocytes and cytoplasm of liver fibroblasts, as well asin pancreatic fibre and in the cytoplasm of pancreatic epithelium. Thus,human and cyno liver and pancreatic tissue exhibit similar stainingpatterns with BRCA68D.

In summary, BRCA84D, BRCA68D, BRCA69D and PRCA157 all showedcross-reactivity in cyno tissues. BRCA84D did not show staining inmonkey liver and pancreas; such staining was observed in human liver andpancreatic tissues. BRCA68D and BRCA69D showed similar stainingintensity and staining patterns in monkey tissues. Although BRCA68D,BRCA69D and PRCA157 showed comparable staining pattern with humantissues, the staining intensity is not identical with human tissues atoptimal conditions. TEST and OVCA22 did not show any staining in monkeytissues at optimal conditions.

A summary of the comparative results of IHC staining in cynomolgustissue and human tissue is provided in Table 14.

TABLE 14 mAb Adrenal Liver Pancreas Kidney Lung Placenta BRCA84DNegative Negative Negative Negative 1/2 Decidual cells 0.625 μg/mlEpithelium 2+ Cynomolgus 1+ Mesenchymal cells negative BRCA84D Negative2/2 Epithelium 1+, Negative Epithelium Decidual cells 0.625 μg/mlSinousoid 5%, 1+, 1+, villi, Human lining cells fibre 2+ 5-10%Mesenchymal 2+, cells 1+ Hepatocytes 1+ 5-10% BRCA68D Cortex 3+ 2/2 1/2Fibroblast Negative Decidual cells, 0.156 μg/ml Hepatocytes Fiber 2+ 1+villi, Cynomolgus 1+ (m) Epithelium 1+ Mesenchymal Sinousoid cells 3+lining cells 1+ BRCA68D Cortex 3+ Sinousoid Epithelium 1+ FibroblastEpithelium Decidual cells 0.156 μg/ml lining cells Fibre 2+ 1+ 1+ 3+,villi, Human 2+, rare Mesenchymal Hepatocytes cells 3+ 2+ (m) BRCA69DCortex 2+ 1/2 1/2 Fibroblast Negative Decidual cells 0.156 μg/mlHepatocytes Epithelium 1+ 1+ 2+, villi, Cynomolgus 1+ (m) rareMesenchymal cells 2+ BRCA69D Cortex 3+ Hepatocytes Epithelium 1+Fibroblast Epithelium Decidual cells 0.156 μg/ml 2+ (m) Fibre 2+ 1+ 1+3+, villi, Human rare Mesenchymal cells 3+ PRCA157 Cortex 1+ 1/2Negative Negative Negative Decidual cells 0.078 μg/ml Hepatocytes 2+,villi, Cynomolgus 1+ (m) Mesenchymal cells 1+ PRCA157 Cortex 2+Sinousoid Epithelium 1+ Negative Negative Not 0.078 μg/ml lining cells5% Determined Human 2+, Fibre 2+ Hepatocytes 1+ (m)

Example 3 B7-H3 mAbs Bind to Multiple ATCC Cancer Cell Lines

The antibodies of the present invention were found to be capable ofbinding to multiple cancer cell lines contained in the collections ofthe American Type Culture Collection. Table 15 and Table 16 summarizethe binding results.

TABLE 15 Antibody Cell Lines BLA08 BRCA68D BRCA69D BRCA84D PRCA157Normal Human Lines HMEC ++/+++ +++ +++ ++ HUVEC ND ++ +/++ +/− +/++Human Breast Cancer Lines BT474 +++ ++ ++/+++ +/++ ++/+++ MCF7 +++ ++++/+++ + ++ MDA175 ND MDA361 ND ++ +/+/− ++ SKBR3 +++ ++ Human LungCancer Lines A549 +++ +/+/− +/− +/− Calu3 +++ +/++ + +/++ SKMES1 +++ ++++/+++ +/++ ++ Human Ovarian Cancer Lines ES-2 +++ +/− SKOV3 +++ ++ +/+++/+/− ++ Human Pancreatic Cancer Lines Panc-1 ++/+++ +/++ +/++ +/+/−+/++ AsPC-1 +++ HPAFII +++ Hs700T +++ ++/+++ +++ +++ Human Colon CancerLines Colo205 ND HT-29 +++ + + + SW480 +++ +/− +/− SW948 ND + + HumanKidney Cancer Lines 293 +++ ++ ++ + ++/+++ 786-0 +++ ++ ++ + ++/+++ A498+++ ++ ++ ++ ++/+++ Caki2 +++ +++ +++ ++ ++/+++ Non-Human Cell LinesCos7 +++ + +/++ +/− +/++ RL65 − SVT2 ND Human Prostate Cancer Lines22Rv1 +++ DU145 +++ + + + +/+/− LNCaP +++ ++ ++ +/++ ++/+++ PC3 ++++/+/− +/− +/− +/− TDH ND +/+/− +/+/− + Human Stomach Cancer Lines HS746TND +/++ +/++ + ++ N87 ND +/++ +/++ +/− +/++

TABLE 16 Antibody Cell Lines TDH06 OVCA22 GB8 SG27 TES7 Normal HumanLines HMEC HUVEC +/+/− +/− +/− Human Breast Cancer Lines BT474 +/++ + +++/++ +/++ MCF7 + + ++ +/+/− + MDA175 ++ MDA361 +/+/− + SKBR3 ++ HumanLung Cancer Lines A549 Calu3 + SKMES1 +/++ +/− +/++ + + Human OvarianCancer Lines ES-2 SKOV3 + +/+/− Human Pancreatic Cancer Lines Panc-1+/+/− + +/− +/+/− AsPC-1 HPAFII + Hs700T + +++ +++ + +++ Human ColonCancer Lines Colo205 + HT-29 + +/+/− +/+/− SW480 +/− +++ SW948 +/− +Human Kidney Cancer Lines 293 +/+/− + +/+/− + 786-0 +  +* +/− + A498 ++/++ +/++ Caki2 ++ + +++ +/++ ++ Non-Human Cell Lines Cos7 + +/+/− * +/−RL65 SVT2 Human Prostate Cancer 22Rv1 + DU145 +/− + LNCaP +/+/− + +*+/+/− + PC3 TDH +++ +/− +/− Human Stomach Cancer Lines HS746T + +/+/−+/− +/− N87 +/+/− +/−

Example 4 B7-H3 mAbs Redirect Killing

The antibodies of the present invention bind to B7-H3 present on thesurface of cancer cells. Using conventional methods, such antibodies maybe labeled with fluorescein, as described above. When such labeledmolecules are incubated in the presence of UDART molecules having anepitope binding domain that binds to the T-cell receptor and an epitopebinding domain that binds to fluorescein (“TCR-UDART”), they can bind tothe DART molecules and thereby localize them to the surface of cellsthat express B7-H3 and cause redirected killing.

A. Redirected Killing of A498 Renal Carcinoma Cells

To demonstrate such redirected killing, fluorescein-labeled B7-H3antibodies were incubated with such TCR-UDART molecules and the abilityof the molecules to mediate cytotoxicity of A498 renal carcinoma cellswas evaluated (Table 17). On the basis of the attained results, the topcandidates were concluded to be: RECA13, BRCA68D, BRCA69D and TDH6.

TABLE 17 Redirected Killing of A498 Renal Carcinoma Cells No With TCR-UDART UDART FACS mAb Mean Mean MFI BCCA66 −1.04 46.39 43.30 BLA8 1.3549.19 50 BRCA165 0 5.11 5.46 BRCA52 0 55.53 41.7 BRCA68D 0 36.89 83.7BRCA69D 0 54.71 84.1 BRCA84D 0 72.40 30.6 GB8 4.00 42.00 17.9 KID1 0.3852.08 18.5 KID13 26.39 58.20 KID35 −1.68 7.62 LUCA1 9.85 52.73 52.9OVCA21 −0.85 47.59 6.04 OVCA22 0.36 38.66 53.9 OVCA25 −2.86 16.70 PA40−0.46 40.54 PRCA123 0 56 130 PRCA135 0 55 127 PRCA157 0 39.14 58.8RECA13 0 38.62 39.8 RECA22 −0.24 51.74 99.90 RECA9 0 62 50.1 SAL3 4.9452.23 60.5 SG24 −2.25 42.00 SG27 −3.98 0.21 SKIN2 3.11 56.44 45.8 STO52.91 37.84 36.7 TDH36 −1.03 53.52 155.00 TDH37 0.05 65.21 47.50 TDH45.09 50.63 45.9 TDH40 −0.65 44.55 TDH5 2.92 49.60 28.8 TDH6 0 70.10 19.5TES7 6.23 52.89 17.5

A498 renal carcinoma cells were incubated with different concentrationsof monoclonal antibodies reactive against B7-H3 in order to determinethe dose-dependent redirected killing mediated by the antibodies. Theresults of the experiments (FIGS. 3A-3B) show that the redirectedkilling was dose-dependent.

B. Redirected Killing of A549 Lung Cancer Cells

To further demonstrate such redirected killing, fluorescein-labeledB7-H3 antibodies were incubated with the above-described TCR-UDARTmolecules or with UDART molecules having an epitope binding domain thatbinds to CD16 and an epitope binding domain that binds to fluorescein(“CD16-UDART”), and the ability of the molecules to mediate cytotoxicityof A549 lung cancer cells was evaluated (Table 18). The results of theexperiments (FIGS. 3C-3D) show that the redirected killing wasdose-dependent. On the basis of the attained results, the top candidateswere concluded to be: BLA8, BRCA68D, BRCA69D and BRCA84D.

TABLE 18 Redirected Killing of A549 Lung Cancer Cells No With TCR- WithCD16- DART UDART UDART FACS mAb Mean Mean Mean MFI BCCA66 1.89 25.178.22 36.1 BLA8 −7.70 10.97 3.68 34.7 BRCA52 0 27.63 37 BRCA68D −4.4213.45 15.95 58.3 BRCA69D 0 24.25 60.5 BRCA84D 0 15.33 25 GB8 −8.68 2.44−4.65 17 KID1 0 22.93 41 LUCA1 0 14.65 53 OVCA21 −2.43 18.90 7.22 31.5OVCA22 0 32.90 61 PRCA123 7.68 29.88 17.31 79.4 PRCA135 −6.58 22.72 8.1475.6 PRCA157 0.02 18.63 18.24 44.3 PSMA −0.70 5.58 9.94 RECA13 0.8617.39 11.90 34.4 RECA22 3.71 20.49 19.35 74.3 RECA9 7.01 26.89 31.8044.3 SAL3 0 31.80 67.4 SKIN2 −0.08 8.65 9.33 41.9 STO5 −10.36 9.28 1.7154.7 TDH36 6.79 24.12 24.08 107 TDH37 6.93 22.57 23.37 42.3 TDH4 −6.2610.07 2.21 32.4 TDH40 4.87 22.01 24.90 53.3 TDH5 −5.08 9.35 −2.85 27.1TDH6 0 19.09 21.3 TES7 0 19.35 15.7C. Redirected Killing of LNcap Prostate Cancer Cells

To further demonstrate such redirected killing, fluorescein-labeledB7-H3 antibodies were incubated with the above-described TCR-UDARTmolecules or with UDART molecules having an epitope binding domain thatbinds to CD16 and an epitope binding domain that binds to fluorescein(“CD16-UDART”), and the ability of the molecules to mediate cytotoxicityof LNcap prostate cancer cells was evaluated (Table 19). On the basis ofthe attained results, the top candidates were concluded to be: BRCA68D,BRCA69D, BRCA84D and PRCA157.

TABLE 19 Redirected Killing of LNcap Prostate Cancer Cells With With NoTCR- CD16- DART UDART UDART FACS mAb Mean Mean Mean MFI BCCA4 −2.9613.29 2.47 5.1 BCCA66 −2.13 13.42 16.40 41 BLA8 4.32 14.97 24.00 48.4BRCA165 3.59 57.26 12.02 7.6 BRCA183D −4.65 43.09 35.30 7.6 BRCA52 32.3471.23 48.28 42.5 BRCA68D −1.40 23.00 21.91 86.9 BRCA69D 40.08 78.0260.55 92.4 BRCA84D 20.11 78.70 41.27 16.4 GB8 −6.25 14.04 10.76 22 KID154.65 91.87 67.86 44.8 KID13 15.86 69.21 47.85 KID133 27.51 45.65 47.12120 KID24 −4.26 34.13 41.17 14.5 KID35 14.17 64.01 33.05 KID47 11.3439.49 15.02 10.8 KID8 16.98 58.80 34.77 5.5 LUCA1 47.40 89.31 67.15 73LUCA17 23.18 26.90 35.87 11.1 LUCAT1 8.25 22.36 21.49 6.9 LUCAT7 26.5038.29 44.77 8.7 MCL12 26.62 35.59 46.38 17.6 MEL2 6.57 29.90 31.40 19OVCA21 12.07 26.81 31.30 41 OVCA22 45.09 96.50 77.30 113 OVCA25 16.1463.26 32.39 PA22 1.73 57.70 9.89 8.9 PA33 8.99 34.49 48.14 9.4 PA4038.42 73.07 63.65 PRCA123 9.96 14.39 18.38 125 PRCA135 −3.75 8.89 13.64123 PRCA157 1.05 17.07 15.43 16.4 PSMA 11.52 31.38 34.79 PSMA 52.8271.19 66.04 RECA13 5.86 22.55 15.40 37 RECA22 7.33 24.65 23.54 22.5RECA9 27.67 52.54 45.14 5.3 SAL1 2.76 17.87 44.52 6.5 SAL2 8.71 30.6829.17 14.5 SAL3 43.79 92.60 76.46 105 SG24 12.64 66.82 44.99 SG27 1.3755.30 16.96 SKIN2 −2.04 14.81 24.23 73.8 SPL16 9.97 29.90 23.74 5.2 STO5−1.48 21.11 24.97 61.3 TDH28 −4.23 18.55 15.04 13.3 TDH36 3.58 19.6119.79 199 TDH37 7.90 18.78 25.22 57.3 TDH4 14.48 37.96 54.64 45.2 TDH408.51 44.55 43.87 79.3 TDH5 7.35 48.71 38.15 29.1 TDH6 4.50 54.59 19.7341.7 TES7 50.15 94.47 73.40 22.4

Example 5 Ability of B7-H3 mAbs to Bind to Soluble B7H3-2Ig and SolubleB7H3-4Ig

As discussed above, B7-H3 exists in both a 4 Ig domain-containing form(B7H3-4Ig) and a 2 Ig domain-containing form (B7H3-2Ig). The anti-B7-H3antibodies of the present invention were tested for their abilities tobind to soluble B7H3-2Ig (FIG. 4A) and soluble B7H3-4Ig B7-H3 (FIG. 4B).The antibodies were found to exhibit a broad range of bindingcharacteristics. Antibodies PRCA123, TDH5, BLA8, BRCA68D and SG24 werefound to exhibit the strongest binding to soluble B7H3-2Ig andantibodies TES7, LUCA50, BRCA165, OVCA22, STO9 and PA20 were found toexhibit the weakest binding to soluble B7H3-2Ig. Antibodies PRCA123,BRCA69A, BLA8 and BRCA68D were found to exhibit the strongest binding tosoluble B7H3-4Ig and antibodies TES7, OVCA21, BRCA165 and STO9 werefound to exhibit the weakest binding to soluble B7H3-4Ig.

Example 6 Affinity Binding of Antigens in Solution to CapturedMonoclonal Antibodies

In order to demonstrate the binding affinity between antigens insolution and captured monoclonal antibodies, antibodies were captured onimmobilized IgG Fc-specific Fab2 fragments at a level of 100-200 RU.Antigens B7-H3 and B7-H3(4Ig) were injected over the captured antibodiesat a concentration of 100 nM (flow rate 20 μl/min for 120 sec, andbinding was measured. Binding responses were normalized to the samelevel of captured mAb and the binding response to m2B6 antibody (mIgG1)control was subtracted as blank. The results of this analysis (FIGS.5A-5S; solid lines; B7-H3(4Ig) 100 nM; dashed lines; B7-H3, 100 nM))demonstrate that the antibodies of the present invention exhibit strongbinding to B7-H3(4Ig).

Example 7 BIACORE™ Analysis: Titration of B7-H3 mAbs to ImmobilizedB7-H3

In order to demonstrate the relative binding affinities of B7-H3-2Ig andB7-H3-4Ig for the antibodies of the present invention, a BIACORE™analysis was performed. B7-H3 antibodies of the present invention werepermitted to bind to immobilized B7-H3-2Ig or to B7-H3-4Ig and thetitration of binding over time was assessed (FIGS. 6A-6I). TDH5,PRCA123, BLA8, BRCA69 were found to have high affinity to both B7-H3-2Igand B7-H3-4Ig. However their epitope(s) were found to be mostly barredin the B7-H3-4Ig molecule, with just a few being available. OVCA22 wasfound to have a very low affinity to both B7-H3-2Ig and B7-H3-4Ig withits epitope being equally available on both molecules. However, it islikely that only the B7-H3-4Ig form provides enough proximity forantibody bivalent binding (low off-rate), whereas the B7-H3-2Ig can bebound only monovalently. TDH6 was found to have barely any affinity inthis format, with binding to 2Ig likely to be non-specific. TES7 andPA20 were found to be B7-H4-4Ig specific antibodies with low affinity.TES7 probably has a low on-rate and a higher off-rate than PA20. BRCA84Dwas found to be an intermediate affinity antibody with a possibility ofmultiple binding sites on both B7-H3-2Ig and B7-H3-4Ig. Based on theBIACORE™ analysis, BRCA84D due to its unusual binding site wasconsidered a preferred antibody. TES7 and PA20 were consideredcandidates for specific binding to high density antigen surfaces, andone of high affinity-low specificity antibody (e.g., BRCA69D oranother).

FIG. 7 provides a comparison BIACORE™ analysis of antibodies PRCA157,BRCA69D, BLA8, PA20, BRCA84D, GB8 and SG27, illustrating that theanti-B7-H3 antibodies of the present invention can exhibit a range ofbinding properties.

FIG. 8 demonstrates the non-competing specificity of several of theanti-B7-H3 antibodies of the present invention. In the experiment, humanB7-H3 molecules were incubated in the presence of antibody BRCA84D andsubjected to BIACORE™ analysis. After approximately 3 minutes a secondanti-B7-H3 antibody was added to the reaction. If the second antibodycompeted with BRCA84D, it would find the B7-H3 sites occluded and beunable to bind. The results indicate that antibodies BRCA68D, BRCA69D,and PRCA157 do not compete with BRCA84D for binding to human B7-H3.

Example 8 Anti-B7-H3 mAbs Internalize on CSC and ATCC Cell Lines

The ability of the anti-B7-H3 antibodies of the present invention tobecome internalized upon binding to cancer cells was investigated.Prostate CSC cells and Hs700t pancreatic cells were incubated with ananti-B7-H3 antibody. The viability of the cells was determined afterincubation in the presence of a saporin-conjugate anti-mouse secondaryantibody which will be toxic to the cells if bound to the primaryantibody and internalized. The results of this investigation forprostate CSC cells (FIG. 9A) and for Hs700t pancreatic cells (FIG. 9B)demonstrate the capacity of the antibodies of the present invention tobecome internalized into cells.

Example 9 B7-H3 mAb Binding and Cross-Blocking Analysis by ELISA

In order to explore the cross-reactivity of the antibodies of thepresent invention and the epitopes recognized by such antibodies, theextent of binding occurring in the presence of a competitor B7-H3antibody was measured. The results of this analysis are shown in FIGS.10A-10F, and show that BRCA68D competes with BRCA69D. TES7 and OVCA22were also found to compete with one another, but TES7 and not OVCA22 wasfound to also compete with both BRCA68D and BRCA69D. GB8 was found tocompete with SG27 for binding to B7-H3-2Ig but not to B7-H3-4Ig. Thedata are summarized in Table 20 and show at least four distinct epitopesfor B7-H3-4Ig (i.e., the epitope recognized by SG27, the epitoperecognized by GB8, the epitope recognized by OVCA22 and TES7, and theepitope recognized by BRCA68D, BRCA69D and TES7) and at least twoepitopes for B7-H3-2Ig (i.e., the epitope recognized by SG27 and GB8,and the epitope recognized by BRCA68D and BRCA69D).

TABLE 20 Summary of B7-H3 mAb Cross-Blocking Analysis by ELISA Antibody(Percent Binding vs. MIgG) Competitor B7-H3 4Ig B7-H3-2Ig Antibody GB8BRCA69D BRCA68D TES7 OVCA22 GB8 BRCA69D BRCA68D GB8 50.211 119.105108.948 87.480 98.142 26.618 84.408 94.710 TES7 111.234 109.390 108.4251.605 16.268 100.645 90.734 99.515 OVCA22 121.783 112.322 100.813 3.3712.048 100.423 87.991 102.766 TDH6 105.591 105.065 100.494 99.839 96.701100.089 66.086 100.728 SG27 101.266 103.021 97.763 78.331 87.789 64.42189.927 94.225 BRCA68D 105.934 40.284 43.144 4.815 102.655 98.888 7.6357.425 BRCA69D 102.558 66.291 71.441 4.334 96.928 94.952 17.346 17.059MIgG 100.000 100.000 100.00 100.000 100.000 100.000 100.000 100.000

The attributes of the key anti-B7-H3 antibodies of the present inventionare shown in Table 21. Based on their exhibited differential staining ofnormal and cancer tissues, their ability to bind B7-H3-4Ig as well asB7-H3-2Ig, their binding affinities as measured by the above-describedBIACORE™ analysis and their ability to bind to cynomolgus B7H3,antibodies, BRCA68D, BRCA69D, BRCA84D, and PRCA157 were judged to be themost preferred antibodies

TABLE 21 MAb BRCA84D TDH6 TES7 BRCA68D BRCA69D GB8 SG27 OVCA22 PRCA157Isotype G1/k G1/k G1/k G1/k G1/k G1/k 2b/k G1/k G1/k IHC 2a 2a 2a 2b 2b2b 2b 2c 2c ATCC Array   2   1 1   3   3 3   1   3   2 Normal TissueColon   1+   1+   1+   1+   2+   2+ Lung   1+   1+   1+ Liver   1+   1+  2+   2+   1+   2+   2+ Kidney   1+   1+   1+   1+ Pancreas   1+   1+  1+   2+ Skin   2+ Cancerous Tissue Colon 1231* 1110* 1.5 2321* 2231*1221* 1122 2231* Lung 1130 1010 1.75 3332 3231 1120 3131** 3231 Prostate 112  111 3  333  333  222  222  333 Breast 1111 1011 3 3333 3333 11223233 2333 Internalization + + + + + + + + + U-DART + + + + + + + + +Specificity 4Ig 4Ig 4Ig 4Ig 4Ig 4Ig 4Ig 4Ig 4Ig 2Ig 2Ig 2Ig 2Ig 2Ig 2Ig2Ig Epitope A B C D D E F G H Group BIACORE ™ + +/− + ++ ++ + + + +/−Cynomolgus ++ + − ++ ++ ++ + + ++ B7-H3 Binding Notes: *Indicatesstaining of stroma **stroma staing 3+

Example 10 Humanized Anti-B7-H3 Antibodies

Monoclonal antibody BRCA84D was humanized in order to produce antibodies(generically designated herein as “hBRCA84D”) offering improved humantherapeutic potential. The sequences of the variable light chain, andthe variable heavy chain, and their respective amino acid andpolynucleotide sequences of a resulting humanized antibody (designatedherein as “hBRCA84D-1”) are provided below:

Humanized BRCA84D-1 Variable Light Chain (SEQ ID NO: 68):DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKPGKAPKLLIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YNNYPFTFGQ GTKLEIK Polynucleotide Sequence Encoding HumanizedBRCA84D-1 Variable Light Chain (SEQ ID NO: 69):gacatccagc tgacccagtc cccctccttc ctgtctgcctccgtgggcga cagagtgacc atcacatgca aggcctcccagaacgtggac accaacgtgg cctggtatca gcagaagcctggcaaggccc ctaagctgct gatctactcc gcctcctaccggtactccgg cgtgccttcc aggttctccg gctccggctctggcaccgac ttcaccctga ccatctccag cctgcagcctgaggacttcg ccacctacta ctgccagcag tacaacaactaccctttcac cttcggccag ggcaccaagc tggaaatcaa gHumanized BRCA84D-1 Variable Light Chain CDR1 (SEQ ID NO: 70):KASQNVDTNVA Polynucleotide Sequence Encoding HumanizedBRCA84D-1 Variable Light Chain CDR₁ (SEQ ID NO: 71):aaggccagtc agaatgtgga tactaatgta gccHumanized BRCA84D-1 Variable Light Chain CDR₂ (SEQ ID NO: 72): SASYRYSPolynucleotide Sequence Encoding HumanizedBRCA84D-1 Variable Light Chain CDR₂ (SEQ ID NO: 73):tcggcatcct accggtacag tHumanized BRCA84D-1 Variable Light Chain CDR₃ (SEQ ID NO: 74): QQYNNYPFTPolynucleotide Sequence Encoding HumanizedBRCA84D-1 Variable Light Chain CDR₃ (SEQ ID NO: 75):cagcaatata acaactatcc attcacg Amino Acid Sequence of Humanized BRCA84D-1Variable Heavy Chain (SEQ ID NO: 80):EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMHWVRQAPGKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDNAKNSLYLQMNSLRDED TAVYYCARGR ENIYYGSRLD YWGQGTTVTV SSPolynucleotide Sequence Encoding HumanizedBRCA84D-1 Variable Heavy Chain (SEQ ID NO: 81):gaggtgcagc tggtcgagtc tggcggagga ctggtgcagcctggcggctc cctgagactg tcttgcgccg cctccggcttcaccttctcc agcttcggca tgcactgggt ccgccaggctccaggcaagg gactggaatg ggtggcctac atctcctccgactcctccgc catctactac gccgacaccg tgaagggcaggttcaccatc tcccgggaca acgccaagaa ctccctgtacctgcagatga actccctgcg ggacgaggac accgccgtgtactactgcgc cagaggccgg gagaatatct actacggctcccggctggat tattggggcc agggcaccac cgtgaccgtg tcctctHumanized BRCA84D-1 Variable Heavy Chain CDR₁ (SEQ ID NO: 82): FGMHPolynucleotide Sequence Encoding HumanizedBRCA84D-1 Variable Heavy Chain CDR₁ (SEQ ID NO: 83): tttggaatgcacHumanized BRCA84D Variable Heavy Chain CDR₂ (SEQ ID NO: 84):YISSDSSAIYYADTVK Polynucleotide Sequence Encoding HumanizedBRCA84D-1 Variable Heavy Chain CDR2 (SEQ ID NO: 85):tacattagta gtgacagtag tgccatctac tatgcagaca cagtgaagHumanized BRCA84D-1 Variable Heavy Chain CDR3 (SEQ ID NO: 86):GRENIYYGSRLDY Polynucleotide Sequence Encoding HumanizedBRCA84D-1 Variable Heavy Chain CDR3 (SEQ ID NO: 87):gggagggaaa acatttacta cggtagtagg cttgactac

FIGS. 11A-11B show the alignment of the amino acid residues of thevariable light chains (FIG. 11A) or variable heavy chains (FIG. 11B) ofBRCA84D and its humanized derivative, hBRCA84D.

In order to obtain hBRCA84D species that exhibit improved affinity forhuman B7-H3, polynucleotides encoding the light or heavy chains ofhBRCA84D-1 (i.e., hBRCA84D-1VL or hBRCA84D-1VH, respectively) weresubjected to mutagenesis, and mutated hBRCA84D-1 light chain derivativeshBRCA84D-2VL, hBRCA84D-3VL, hBRCA84D-4VL, hBRCA84D-5VL, and hBRCA84D-6VLand mutated hBRCA84D-1 heavy chain derivatives hBRCA84D-2VH,hBRCA84D-3VH, and hBRCA84D-4VH were isolated and characterized. Theamino acid and polynucleotide sequences of the variable light and heavychains of these antibodies are presented below:

hBRCA84D-2VL (SEQ ID NO: 89):DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKPGKAPKALIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YNNYPFTFGQ GTKLEIKPolynucleotide Encoding hBRCA84D-2VL (SEQ ID NO: 90):gacatccagc tgacccagtc cccctccttc ctgtctgcctccgtgggcga cagagtgacc atcacatgca aggcctcccagaacgtggac accaacgtgg cctggtatca gcagaagcctggcaaggccc ctaaggcgct gatctactcc gcctcctaccggtactccgg cgtgccttcc aggttctccg gctccggctctggcaccgac ttcaccctga ccatctccag cctgcagcctgaggacttcg ccacctacta ctgccagcag tacaacaactaccctttcac cttcggccag ggcaccaagc tggaaatcaa ghBRCA84D-3VL (SEQ ID NO: 91):DIQLTQSPSF LSASVGDRVS VTCKASQNVD TNVAWYQQKPGKAPKLLIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YNNYPFTFGQ GTKLEIKPolynucleotide Encoding hBRCA84D-3VL (SEQ ID NO: 92):gacatccagc tgacccagtc cccctccttc ctgtctgcctccgtgggcga cagagtgtcc gtcacatgca aggcctcccagaacgtggac accaacgtgg cctggtatca gcagaagcctggcaaggccc ctaagctgct gatctactcc gcctcctaccggtactccgg cgtgccttcc aggttctccg gctccggctctggcaccgac ttcaccctga ccatctccag cctgcagcctgaggacttcg ccacctacta ctgccagcag tacaacaactaccctttcac cttcggccag ggcaccaagc tggaaatcaa ghBRCA84D-4VL (SEQ ID NO: 93):DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKPGQAPKLLIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YNNYPFTFGQ GTKLEIKPolynucleotide Encoding hBRCA84D-4VL (SEQ ID NO: 94):gacatccagc tgacccagtc cccctccttc ctgtctgcctccgtgggcga cagagtgacc atcacatgca aggcctcccagaacgtggac accaacgtgg cctggtatca gcagaagcctggccaggccc ctaagctgct gatctactcc gcctcctaccggtactccgg cgtgccttcc aggttctccg gctccggctctggcaccgac ttcaccctga ccatctccag cctgcagcctgaggacttcg ccacctacta ctgccagcag tacaacaactaccctttcac cttcggccag ggcaccaagc tggaaatcaa ghBRCA84D-5VL (SEQ ID NO: 95):DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKPGQAPKALIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YNNYPFTFGQ GTKLEIKPolynucleotide Encoding hBRCA84D-5VL (SEQ ID NO: 96):gacatccagc tgacccagtc cccctccttc ctgtctgcctccgtgggcga cagagtgacc atcacatgca aggcctcccagaacgtggac accaacgtgg cctggtatca gcagaagcctggccaggccc ctaaggcgct gatctactcc gcctcctaccggtactccgg cgtgccttcc aggttctccg gctccggctctggcaccgac ttcaccctga ccatctccag cctgcagcctgaggacttcg ccacctacta ctgccagcag tacaacaactaccctttcac cttcggccag ggcaccaagc tggaaatcaa ghBRCA84D-6VL (SEQ ID NO: 97):DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKPGKAPKLLIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQPEDFAEYYCQQ YNNYPFTFGQ GTKLEIKPolynucleotide Encoding hBRCA84D-6VL (SEQ ID NO: 98):gacatccagc tgacccagtc cccctccttc ctgtctgcctccgtgggcga cagagtgacc atcacatgca aggcctcccagaacgtggac accaacgtgg cctggtatca gcagaagcctggcaaggccc ctaagctgct gatctactcc gcctcctaccggtactccgg cgtgccttcc aggttctccg gctccggctctggcaccgac ttcaccctga ccatctccag cctgcagcctgaggacttcg ccgagtacta ctgccagcag tacaacaactaccctttcac cttcggccag ggcaccaagc tggaaatcaa ghBRCA84D-2VH (SEQ ID NO: 99):EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMHWVRQAPGKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDNAKNSLYLQMNSLRDED TAVYYCGRGR ENIYYGSRLD YWGQGTTVTV SSPolynucleotide Encoding hBRCA84D-2VH (SEQ ID NO: 100):gaggtgcagc tggtcgagtc tggcggagga ctggtgcagcctggcggctc cctgagactg tcttgcgccg cctccggcttcaccttctcc agcttcggca tgcactgggt ccgccaggctccaggcaagg gactggaatg ggtggcctac atctcctccgactcctccgc catctactac gccgacaccg tgaagggcaggttcaccatc tcccgggaca acgccaagaa ctccctgtacctgcagatga actccctgcg ggacgaggac accgccgtgtactactgcgg cagaggccgg gagaatatct actacggctcccggctggat tattggggcc agggcaccac cgtgaccgtg tcctcthBRCA84D-3VH (SEQ ID NO: 101):EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMHWVRQAPGKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDNAKNSLYLQMNSLRDED TAMYYCGRGR ENIYYGSRLD YWGQGTTVTV SSPolynucleotide Encoding hBRCA84D-3VH (SEQ ID NO: 102):gaggtgcagc tggtcgagtc tggcggagga ctggtgcagcctggcggctc cctgagactg tcttgcgccg cctccggcttcaccttctcc agcttcggca tgcactgggt ccgccaggctccaggcaagg gactggaatg ggtggcctac atctcctccgactcctccgc catctactac gccgacaccg tgaagggcaggttcaccatc tcccgggaca acgccaagaa ctccctgtacctgcagatga actccctgcg ggacgaggac accgccatgtactactgcgg cagaggccgg gagaatatct actacggctcccggctggat tattggggcc agggcaccac cgtgaccgtg tcctcthBRCA84D-4VH (SEQ ID NO: 103):EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMHWVRQAPGKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDNAKNSLYLQMNSLRSED TAVYYCARGR ENIYYGSRLD YWGQGTTVTV SSPolynucleotide Encoding hBRCA84D-4VH (SEQ ID NO: 104):gaggtgcagc tggtcgagtc tggcggagga ctggtgcagcctggcggctc cctgagactg tcttgcgccg cctccggcttcaccttctcc agcttcggca tgcactgggt ccgccaggctccaggcaagg gactggaatg ggtggcctac atctcctccgactcctccgc catctactac gccgacaccg tgaagggcaggttcaccatc tcccgggaca acgccaagaa ctccctgtacctgcagatga actccctgcg gagcgaggac accgccgtgtactactgcgc cagaggccgg gagaatatct actacggctcccggctggat tattggggcc agggcaccac cgtgaccgtg tcctct

Table 22 lists the hBRCA84D variable light chain and variable heavychain mutations studied; numbers refer to the Kabat numbering systemused in FIGS. 11A and 11B.

TABLE 22 Variable Light Chain Variable Heavy Chain Kabat Position 20 2142 46 85 Kabat Position 84 89 93 BRCA84D S V Q A E BRCA84D S M GhBRCA84D-1VL T I K L T hBRCA84D-1VH D V A hBRCA84D-2VL T I K A ThBRCA84D-2VH D V G hBRCA84D-3VL S V K L T hBRCA84D-3VH D M GhBRCA84D-4VL T I Q L T hBRCA84D-4VH S V A hBRCA84D-5VL T I Q A ThBRCA84D-6VL T I K L E

The relative binding affinities of the hBRCA84D light chain derivativeshBRCA84D-3VL, hBRCA84D-4VL and hBRCA84D-5VL for human B7-H3 weredetermined by forming antibodies containing these light chain variableregions and a chimeric BRCA84D-1VH heavy chain (FIG. 12). BRCA84D-5VL(K42Q, L46A) was found to have the highest binding affinity of thehBRCA84D-VL tested. BRCA84D-5VL was therefore used as the light chain toinvestigate the relative binding affinities of the hBRCA84D heavy chainsof hBRCA84D-1VH, hBRCA84D-2VH, hBRCA84D-3VH and hBRCA84D-4VH for humanB7-H3 (FIG. 13). hBRCA84D-2VH (A93G) was found to have the highestbinding affinity of the hBRCA84D-VH tested.

The amino acid and encoding polynucleotide sequences of the chimericBRCA84D-1 are as follows:

chBRCA84D Light Chain (SEQ ID NO: 105):DIAMTQSQKF MSTSVGDRVS VTCKASQNVD TNVAWYQQKPGQSPKALIYS ASYRYSGVPD RFTGSGSGTD FTLTINNVQSEDLAEYFCQQ YNNYPFTFGS GTKLEIKRTV AAPSVFIFPPSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGECPolynucleotide Encoding chBRCA84D Light Chain (SEQ ID NO: 106):gacattgcga tgacccagtc tcaaaaattc atgtccacatcagtaggaga cagggtcagc gtcacctgca aggccagtcagaatgtggat actaatgtag cctggtatca acagaaaccagggcaatctc ctaaagcact gatttactcg gcatcctaccggtacagtgg agtccctgat cgcttcacag gcagtggatctgggacagat ttcactctca ccatcaacaa tgtgcagtctgaagacttgg cagagtattt ctgtcagcaa tataacaactatccattcac gttcggctcg gggacaaagt tggaaataaaacgtacggtg gctgcaccat ctgtcttcat cttcccgccatctgatgagc agttgaaatc tggaactgcc tctgttgtgtgcctgctgaa taacttctat cccagagagg ccaaagtacagtggaaggtg gataacgccc tccaatcggg taactcccaggagagtgtca cagagcagga cagcaaggac agcacctacagcctcagcag caccctgacg ctgagcaaag cagactacgagaaacacaaa gtctacgcct gcgaagtcac ccatcagggcctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttagchBRCA84D Heavy Chain (SEQ ID NO: 107):DVQLVESGGG LVQPGGSRKL SCAASGFTFS SFGMHWVRQAPEKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDNPKNTLFLQMTSLRSED TAMYYCGRGR ENIYYGSRLD YWGQGTTLTVSSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVTVSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTQTYICNVNHK PSNTKVDKRV EPKSCDKTHT CPPCPAPELLGGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSRDELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKPolynucleotide Encoding chBRCA84D Heavy Chain (SEQ ID NO: 108):gatgtgcagc tggtggagtc tgggggaggc ttagtgcagcctggagggtc ccggaaactc tcctgtgcag cctctggattcactttcagt agctttggaa tgcactgggt tcgtcaggctccagagaagg ggctggagtg ggtcgcatac attagtagtgacagtagtgc catctactat gcagacacag tgaagggccgattcaccatc tccagagaca atcccaagaa caccctgttcctgcaaatga ccagtctaag gtctgaggac acggccatgtattactgtgg aagagggagg gaaaacattt actacggtagtaggcttgac tactggggcc aaggcaccac tctcacagtctcctcagcct ccaccaaggg cccatcggtc ttccccctggcaccctcctc caagagcacc tctgggggca cagcggccctgggctgcctg gtcaaggact acttccccga accggtgacggtgtcgtgga actcaggcgc cctgaccagc ggcgtgcacaccttcccggc tgtcctacag tcctcaggac tctactccctcagcagcgtg gtgaccgtgc cctccagcag cttgggcacccagacctaca tctgcaacgt gaatcacaag cccagcaacaccaaggtgga caagagagtt gagcccaaat cttgtgacaaaactcacaca tgcccaccgt gcccagcacc tgaactcctggggggaccgt cagtcttcct cttcccccca aaacccaaggacaccctcat gatctcccgg acccctgagg tcacatgcgtggtggtggac gtgagccacg aagaccctga ggtcaagttcaactggtacg tggacggcgt ggaggtgcat aatgccaagacaaagccgcg ggaggagcag tacaacagca cgtaccgtgtggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaatggcaaggagt acaagtgcaa ggtctccaac aaagccctcccagcccccat cgagaaaacc atctccaaag ccaaagggcagccccgagaa ccacaggtgt acaccctgcc cccatcccgggatgagctga ccaagaacca ggtcagcctg acctgcctggtcaaaggctt ctatcccagc gacatcgccg tggagtgggagagcaatggg cagccggaga acaactacaa gaccacgcctcccgtgctgg actccgacgg ctccttcttc ctctacagcaagctcaccgt ggacaagagc aggtggcagc aggggaacgtcttctcatgc tccgtgatgc atgaggctct gcacaaccactacacgcaga agagcctctc cctgtctccg ggtaaatgaAmino Acid Sequence Of The hBRCA84D-2 Light Chain (SEQ ID NO: 117):DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKPGKAPKALIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YNNYPFTFGQ GTKLEIKRTV AAPSVFIFPPSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGECPolynucleotide Encoding The Amino Acid Sequence OfThe hBRCA84D-2 Light Chain (SEQ ID NO: 118):gacatccagc tgacccagtc cccctccttc ctgtctgcctccgtgggcga cagagtgacc atcacatgca aggcctcccagaacgtggac accaacgtgg cctggtatca gcagaagcctggcaaggccc ctaaggcgct gatctactcc gcctcctaccggtactccgg cgtgccttcc aggttctccg gctccggctctggcaccgac ttcaccctga ccatctccag cctgcagcctgaggacttcg ccacctacta ctgccagcag tacaacaactaccctttcac cttcggccag ggcaccaagc tggaaatcaagcgtacggtg gctgcaccat ctgtcttcat cttcccgccatctgatgagc agttgaaatc tggaactgcc tctgttgtgtgcctgctgaa taacttctat cccagagagg ccaaagtacagtggaaggtg gataacgccc tccaatcggg taactcccaggagagtgtca cagagcagga cagcaaggac agcacctacagcctcagcag caccctgacg ctgagcaaag cagactacgagaaacacaaa gtctacgcct gcgaagtcac ccatcagggcctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttagAmino Acid Sequence Of The hBRCA84D-2 Heavy Chain (SEQ ID NO: 119):EVQLVESGGG LVQPGGSLRL SCAASGFTFS SFGMHWVRQAPGKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDNAKNSLYLQMNSLRDED TAVYYCGRGR ENIYYGSRLD YWGQGTTVTVSSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVTVSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGTQTYICNVNHK PSNTKVDKRV EPKSCDKTHT CPPCPAPELVGGPSVFLLPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPPEEQ YNSTLRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSREEMTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPLVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKPolynucleotide Encoding The Amino Acid Sequence OfThe hBRCA84D-2 Heavy Chain (SEQ ID NO: 120):gaggtgcagc tggtcgagtc tggcggagga ctggtgcagcctggcggctc cctgagactg tcttgcgccg cctccggcttcaccttctcc agcttcggca tgcactgggt ccgccaggctccaggcaagg gactggaatg ggtggcctac atctcctccgactcctccgc catctactac gccgacaccg tgaagggcaggttcaccatc tcccgggaca acgccaagaa ctccctgtacctgcagatga actccctgcg ggacgaggac accgccgtgtactactgcgg cagaggccgg gagaatatct actacggctcccggctggat tattggggcc agggcaccac cgtgaccgtgtcctctgcct ccaccaaggg cccatcggtc ttccccctggcaccctcctc caagagcacc tctgggggca cagcggccctgggctgcctg gtcaaggact acttccccga accggtgacggtgtcgtgga actcaggcgc cctgaccagc ggcgtgcacaccttcccggc tgtcctacag tcctcaggac tctactccctcagcagcgtg gtgaccgtgc cctccagcag cttgggcacccagacctaca tctgcaacgt gaatcacaag cccagcaacaccaaggtgga caagagagtt gagcccaaat cttgtgacaaaactcacaca tgcccaccgt gcccagcacc tgaactcgtggggggaccgt cagtcttcct cttaccccca aaacccaaggacaccctcat gatctcccgg acccctgagg tcacatgcgtggtggtggac gtgagccacg aagaccctga ggtcaagttcaactggtacg tggacggcgt ggaggtgcat aatgccaagacaaagccgcc ggaggagcag tacaacagca cgctccgtgtggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaatggcaaggagt acaagtgcaa ggtctccaac aaagccctcccagcccccat cgagaaaacc atctccaaag ccaaagggcagccccgagaa ccacaggtgt acaccctgcc cccatcccgggaggagatga ccaagaacca ggtcagcctg acctgcctggtcaaaggctt ctatcccagc gacatcgccg tggagtgggagagcaatggg cagccggaga acaactacaa gaccacgcctctcgtgctgg actccgacgg ctccttcttc ctctacagcaagctcaccgt ggacaagagc aggtggcagc aggggaacgtcttctcatgc tccgtgatgc atgaggctct gcacaaccactacacgcaga agagcctctc cctgtctccg ggtaaa

The relative binding affinities of antibodies containing: (1)hBRCA84D-2VL and hBRCA84D-2VH (two trials), (2) chimeric BRCA84D, (3)antibody containing hBRCA84D-5VL and chimeric BRCA84D-HC, and (4)antibody containing hBRCA84D-5VL and hBRCA84D-2VH were compared. Theresults are shown in FIG. 14.

Example 11 Humanized Anti-B7-H3 Antibodies Inhibit Tumor Growth inXenografts

In order to demonstrate the ability of humanized anti-B7-H3 antibodiesto inhibit tumor growth in vivo, tumor growth of HT-1197 urinary bladdercarcinoma cells and of A498 renal carcinoma cells was studied in amurine xenograft. Humanized antibody hBRCA84D-2 (hBRCA84D-2 VLchain/hBRCA84D-2 VL chain) was modified to comprise an Fc region havingsubstitutions L235V, F243L, R292P, Y300L, and P396L.

The Fc-modified hBRCA84D-2 antibody was administered to the mice (at adose of 1 μg/kg, 10 μg/kg, or 20 μg/kg) 7 days, 14 days, 21 days and 28days post implantation of the cancer cells. The results show that at alldoses the administered Fc-modified hBRCA84D-2 antibody was capable ofinhibiting tumor growth of HT-1197 urinary bladder carcinoma cells (FIG.15) and of A498 renal carcinoma cells (FIG. 16).

Example 12 Dual Affinity Retargeting Reagents (DARTs) Specific for B7-H3and the T-Cell Receptor Mediate Potent Redirected T-Cell Killing

Dual affinity retargeting reagents (DARTs) specific for B7-H3 and theT-cell receptor (“TCR”) and for the Natural Killer Group 2D (NKG2D)receptor were prepared. Such DARTs have the ability to localize a T-cell(by binding such T-cell to the TCR-binding portion of a TCR-bindingDART) or o localize a NK-cell (by binding such NK cell to theNKG2D-binding portion of an NKG2D-binding DART) to the location of acancer cell (by binding such cancer cell to the B7-H3-binding portion ofthe DART). The localized T-cell or NK cell can then mediate the killingof the cancer cell in a process termed herein “redirected” killing.

The dual affinity retargeting reagent (DART) specific for B7-H3 and theT-cell receptor (“TCR”) was constructed having the anti-B7-H3 variabledomains of hBRCA84D-2 and anti-TCR variable domains:

TCR VL × hBRCA84D VH-2-E Coil DART Chain (SEQ ID NO: 109):EIVLTQSPAT LSLSPGERAT LSCSATSSVS YMHWYQQKPGKAPKRWIYDT SKLASGVPSR FSGSGSGTEF TLTISSLQPE DFATYYCQQW SSNPLTFGQG TKLEIKGGGS   GGGG EVQLVE SGGGLVQPGG SLRLSCAASG FTFSSFGMHW VRQAPGKGLEWVAYISSDSS AIYYADTVKG RFTISRDNAK NSLYLQMNSLRDEDTAVYYC GRGRENIYYG SRLDYWGQGT TVTVSS GGCGGGEVAALEKE VAALEKEVAA LEKEVAALEK  Polynucleotide Encoding TCR VL ×hBRCA84D VH-2-E Coil DART Chain (SEQ ID NO: 110):gaaattgtgt tgacacagtc tccagccacc ctgtctttgtctccagggga aagagccacc ctctcctgca gtgccacctcaagtgtaagt tacatgcact ggtatcagca gaaaccagggaaagccccta agcgctggat ctatgacaca tccaaactggcttctggggt cccatcaagg ttcagcggca gtggatctgggacagaattt actctcacaa tcagcagcct gcagcctgaagattttgcaa cttattactg tcagcagtgg agtagtaacccgctcacgtt tggccagggg accaagcttg agatcaaa gg aggcggatcc   ggcggcggag  gc gaggtgca gctggtcgag tctggcggag gactggtgca gcctggcggc tccctgagactgtcttgcgc cgcctccggc ttcaccttct ccagcttcggcatgcactgg gtccgccagg ctccaggcaa gggactggaatgggtggcct acatctcctc cgactcctcc gccatctactacgccgacac cgtgaagggc aggttcacca tctcccgggacaacgccaag aactccctgt acctgcagat gaactccctgcgggacgagg acaccgccgt gtactactgc ggcagaggccgggagaatat ctactacggc tcccggctgg attattggggccagggcacc accgtgaccg tgtcctcc gg   aggatgtggcggtggagaag tggccgcact ggagaaagag gttgctgctttggagaagga ggtcgctgca cttgaaaagg aggtcgcagc cctggagaaa hBRCA84DVL-2 ×TCR VH-K coil Chain (SEQ ID NO: 111):DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKPGKAPKALIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YNNYPFTFGQ GTKLEIK GGG   SGGGG QVQLVQSGAEVKKPG ASVKVSCKAS GYKFTSYVMH WVRQAPGQGLEWIGYINPYN DVTKYNEKFK GRVTITADKS TSTAYLQMNSLRSEDTAVHY CARGSYYDYD GFVYWGQGTL VTVSS GGCGGGKVAALKEKV AALKEKVAAL KEKVAALKE Polynucleotide Encoding hBRCA84DVL-2 ×TCR VH - K coil Chain (SEQ ID NO: 112):gacatccagc tgacccagtc cccctccttc ctgtctgcctccgtgggcga cagagtgacc atcacatgca aggcctcccagaacgtggac accaacgtgg cctggtatca gcagaagcctggcaaggccc ctaaggcgct gatctactcc gcctcctaccggtactccgg cgtgccttcc aggttctccg gctccggctctggcaccgac ttcaccctga ccatctccag cctgcagcctgaggacttcg ccacctacta ctgccagcag tacaacaactaccctttcac cttcggccag ggcaccaagc tggaaatcaa g ggaggcgga   tccggcggcg  gaggc caggt tcagctggtg cagtctggag ctgaggtgaa gaagcctggg gcctcagtgaaggtctcctg caaggccagc ggttacaagt ttaccagctacgtgatgcac tgggtgcgac aggcccctgg acaagggcttgagtggatcg gatatattaa tccttacaat gatgttactaagtacaatga gaagttcaaa ggcagagtca cgattaccgcggacaaatcc acgagcacag cctacctgca gatgaacagcctgagatccg aggacacggc cgtgcactac tgtgcgagagggagctacta tgattacgac gggtttgttt actggggcca agggactctg gtcactgtga gctccggagg   atgtggcggt ggaaaagtgg ccgcactgaa ggagaaagtt gctgctttgaaagagaaggt cgccgcactt aaggaaaagg tcgcagccct gaaagag

The dual affinity retargeting reagent (DART) specific for B7-H3 and theNatural Killer Group 2D (NKG2D) receptor was constructed having theanti-B7-H3 variable domains of hBRCA84D-2 and anti-TCR variable domains:

NKG2D VL × hBRCA84D VH-2-E Coil DART Chain (SEQ ID NO: 113):QSALTQPASV SGSPGQSITI SCSGSSSNIG NNAVNWYQQLPGKAPKLLIY YDDLLPSGVS DRFSGSKSGT SAFLAISGLQSEDEADYYCA AWDDSLNGPV FGGGTKLTVL  GGGSGGGG EVQLVESGGGLV QPGGSLRLSC AASGFTFSSF GMHWVRQAPGKGLEWVAYIS SDSSAIYYAD TVKGRFTISR DNAKNSLYLQMNSLRDEDTA VYYCGRGREN IYYGSRLDYW GQGTTVTVSSGGCGGGEVAA LEKEVAALEKE VAALEKEVA ALEK Polynucleotide Encoding NKG2D VL ×hBRCA84D VH-2- E Coil DART Chain (SEQ ID NO: 114):cagtctgccc tgactcagcc tgcctccgtg tctgggtctcctggacagtc aatcaccatc tcctgttctg gaagcagctccaacatcgga aataatgctg ttaactggta ccagcagctcccaggaaagg ctcccaaact cctcatctat tatgatgacctactgccctc aggggtctct gaccgattct ctggctccaagtctggcacc tcagccttcc tggccatcag tgggctccagtctgaggatg aggctgatta ttactgtgca gcatgggatgacagcctgaa tggtccagtg ttcggcggag ggaccaagct gaccgtccta  ggaggcggat  ccggcggcgg   aggc gaggtg cagctggtcg agtctggcgg aggactggtg cagcctggcggctccctgag actgtcttgc gccgcctccg gcttcaccttctccagcttc ggcatgcact gggtccgcca ggctccaggcaagggactgg aatgggtggc ctacatctcc tccgactcctccgccatcta ctacgccgac accgtgaagg gcaggttcaccatctcccgg gacaacgcca agaactccct gtacctgcagatgaactccc tgcgggacga ggacaccgcc gtgtactactgcggcagagg ccgggagaat atctactacg gctcccggctggattattgg ggccagggca ccaccgtgac cgtgtcctcc ggaggatgtg  gcggtggaga agtggccgca ctggagaaagaggttgctgc tttggagaag gaggtcgctg cacttgaaaa ggaggtcgca gccctggaga aahBRCA84DVL-2 × NKG2D VH - K coil Chain (SEQ ID NO: 115):DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKPGKAPKALIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQPEDFATYYCQQ YNNYPFTFGQ GTKLEIK GGG   SGGGG QVQLVESGGGLVKPG GSLRLSCAAS GFTFSSYGMH WVRQAPGKGLEWVAFIRYDG SNKYYADSVK GRFTISRDNS KNTLYLQMNSLRAEDTAVYY CAKDRGLGDG TYFDYWGQGT TVTVSS GGCGGGKVAALKEK VAALKEKVAA LKEKVAALKE Polynucleotide Encoding hBRCA84DVL-2 ×NKG2D VH - K coil Chain (SEQ ID NO: 116):gacatccagc tgacccagtc cccctccttc ctgtctgcctccgtgggcga cagagtgacc atcacatgca aggcctcccagaacgtggac accaacgtgg cctggtatca gcagaagcctggcaaggccc ctaaggcgct gatctactcc gcctcctaccggtactccgg cgtgccttcc aggttctccg gctccggctctggcaccgac ttcaccctga ccatctccag cctgcagcctgaggacttcg ccacctacta ctgccagcag tacaacaactaccctttcac cttcggccag ggcaccaagc tggaaatcaa g ggaggcgga   tccggcggcg  gaggc caggt acagctggtg gagtctgggg gaggcctggt caagcctgga gggtccctgagactctcctg tgcagcgtct ggattcacct tcagtagctatggcatgcac tgggtccgcc aggctccagg caaggggctggagtgggtgg catttatacg gtatgatgga agtaataaatactatgcaga ctccgtgaag ggccgattca ccatctccagagacaattcc aagaacacgc tgtatctgca aatgaacagcctgagagctg aggacacggc tgtgtattac tgtgcgaaagatcgaggttt gggggatgga acctactttg actactggggccaagggacc acggtcaccg tctcctcc gg   aggatgtggcggtggaaaag tggccgcact gaaggagaaa gttgctgctttgaaagagaa ggtcgccgca cttaaggaaa aggtcgcagc cctgaaagag

In order to demonstrate the ability of DARTs to mediate such redirectedkilling of cancer cells, the above-described hBRCA84D-2/anti-TCR DART(“T-DART”), hBRCA84D-2, hBRCA84D-2 (Fc-modified: L235V, F243L, R292P,Y300L, and P396L), and a TCR-DART control were incubated at variousconcentrations with target cancer cells (SK-MES-1 lung cancer cells,A498 renal carcinoma cells, LNCaP prostate cancer cells, or UACC-62melanoma cells) and effector resting PBMC (E:T ratio=30:1) andcytotoxicity was determined (LDH Assay). The results of theseinvestigations are shown in FIGS. 17A-17D and demonstrate the ability ofthe hBRCA84D-2/anti-TCR DART (“T-DART”) to mediate redirected killing ofcancer cells.

Example 13 Pharmacokinetic Profile in Tumor-Free Mice

Anti-B7-H3 antibody (Mab1) was injected into male mCD16−/−, hCD16A_FOXN1mice (5 mg/kg; IV) and serum was assayed (pre-dose and) at 2, 15, 30min, and 1, 2, 4, 6 hr, and 1, 2, 3, 6, 8, 14, 21, and 28 days afterinjection. The antibody was found to have a T ½ of 10.54 days and aC_(max) of 43.493 μg/ml. The concentration of antibody over time wasfound to be biphasic, fitting into a two-component model (FIGS.18A-18B). The predicted pharmacokinetic profiles generated using a2-compartment model with parameters from the 5 mg/kg dose are shown inFIG. 18C.

Example 14 Ability of Anti-B7-H3 Antibody to Bind HT-1197 UrinaryBladder Cancer Cells and Prevent or Inhibit Tumor Development in aMurine Xenograft Model

The above-described anti-B7-H3 antibody (Mab1) was assessed for itsability to bind HT-1197, a human B7-H3-expressing urinary bladdercarcinoma cell line. As shown in FIG. 19, such cells exhibit greaterexpression of PRCA135 than HER2, and thus are particularly suitable forassessing the therapeutic potential of the antibodies of the presentinvention in remediating HT-1197 tumors. In accordance with thisconclusion, anti-B7-H3 antibody hBRCA84D variants were found to becapable of binding to HT-1197 cells. FIG. 20 shows the binding affinityof Mab1 antibodies to HT-1197 cells.

Mice (mCD16−/−, hCD16A+_FoxN1) were implanted subcutaneously on theirflanks with 8×10⁶ HT-1197 cells. The tumor cells were implanted in 200μl of Ham's F12 Medium diluted 1:1 with MATRIGEL™. Treatment with Mab1was initiated within 7 days of implantation via iv Q7D×5 using doses of0.1, 0.5, 1, 5, or 10 mg/kg (eight female mice per dose). Centuximab(anti-EGRF antibody) was administered to a control group of mice atdoses of 1, 5, or 15 mg/kg (eight female mice per dose). Eight femalemice were also injected with vehicle or with 10 mg/kg IgG control. Tumormeasurements were made every 3-4 days. The results of the experiment(FIG. 21A) show that Mab1 was capable of preventing or inhibitingurinary bladder tumor development in the murine xenograft model. FIG.21B shows the results obtained using centimab. A comparison of FIGS. 21Aand 21B demonstrate that the antibodies of the present invention aremore effective than centimab in preventing or inhibiting urinary bladdertumor development in the murine xenograft model. FIG. 21C compares theresults obtained at the maximum doses tested.

Example 15 Ability of Anti-B7-H3 Antibody to Bind HT-1376 UrinaryBladder Cancer Cells and Prevent or Inhibit Tumor Development in aMurine Xenograft Model

The above-described anti-B7-H3 antibody (Mab1) was assessed for itsability to bind HT-1376, a human B7-H3-expressing urinary bladdercarcinoma cell line. As shown in FIGS. 22A-22B, such cells exhibitgreater expression of PRCA135 than HER2 or PMSA, and thus areparticularly suitable for assessing the therapeutic potential of theantibodies of the present invention in remediating HT-1376 tumors. Inaccordance with this conclusion, anti-B7-H3 antibody hBRCA84D variantswere found to be capable of binding to HT-1376 cells. FIGS. 22A-22B showthe binding affinity of Mab1 antibodies to HT-1376 cells.

Mice (mCD16−/−, hCD16A+_FoxN1) were implanted subcutaneously on theirflanks with 5×10⁶ HT-1376 cells. The tumor cells were implanted in 200μl of Ham's F12 Medium diluted 1:1 with MATRIGEL™. Treatment with Mab1was initiated within 7 days of implantation via iv Q7D×4 at a dose of 1mg/kg. The results of the experiment (FIG. 23) show that Mab1 wascapable of preventing or inhibiting urinary bladder tumor development inthe murine xenograft model.

Example 16 Ability of Anti-B7-H3 Antibody to Bind Cancer Cells

Anti-B7-H3 antibody BRCA84D was assessed via FACS analysis for itsability to bind: SW480 and SW620 colorectal cancer cells; AGS gastriccancer cells; M-14 and LOX IMVI melanoma cells; 22rv prostate cancercells; AsPC-1 and BxPc-3 pancreatic cancer cells; A498 and 786-0 renalcancer cells. The antibody was found to be able to bind to all suchcells.

Example 17 Ability of Anti-B7-H3 Antibody Prevent or Inhibit GastricTumor Development in a Murine Xenograft Model

Mice (mCD16−/−, hCD16A+_FoxN1) were implanted subcutaneously on theirflanks with 5×10⁶ AGS cells. The tumor cells were implanted in 200 μl ofHam's F12 Medium diluted 1:1 with MATRIGEL™. Treatment with Mab1 wasinitiated within 7 days of implantation via iv Q7D×5 using doses of 0.5,1, 5, or 10 mg/kg. The results of the experiment (FIG. 24) show thatMab1 was capable of preventing or inhibiting gastric tumor developmentin the murine xenograft model.

Example 18 Ability of Anti-B7-H3 Antibody to Bind Lung Cancer Cells andPrevent or Inhibit Tumor Development in a Murine Xenograft Model

A549 lung cancer cells were incubated in the presence of hBRCA84D,chBRCA84D and hBRCA84 (0264 Fc) variant and the cytotoxic effect ofthese antibodies was determined. The results of this experiment areshown in FIG. 25, and indicate that all three of the antibodies werecytotoxic to A549 cells.

Mice (mCD16−/−, hCD16A+_FoxN1) were implanted subcutaneously on theirflanks with 8×10⁶ A549 cells. The tumor cells were implanted in 200 μlof Ham's F12 Medium diluted 1:1 with MATRIGEL™. Treatment with Mab1 wasinitiated within 7 days of implantation via iv Q7D×4 using a dose of 1mg/kg. The results of the experiment (FIG. 26) show that Mab1 wascapable of preventing or inhibiting of lung cancer tumor development inthe murine xenograft model.

FACS analysis was conducted on CaLu3 lung cancer cells in order todetermine whether such cells bind anti-B7-H3 antibodies. The experimentconfirmed that such cells express B7-H3 and bind to the antibodies ofthe present invention. To determine whether the antibodies of thepresent invention were effective to prevent or inhibit lung cancer tumordevelopment, mice (mCD16−/−, hCD16A+_FoxN1) were implantedsubcutaneously on their flanks with 5×10⁶ CaLu3 cells. The tumor cellswere implanted in 200 μl of Ham's F12 Medium diluted 1:1 with MATRIGEL™.Treatment with Mab1 was initiated within 7 days of implantation via ivQ7D×4 using a dose of 0.5, 1, or 5 mg/kg. The results of the experiment(FIG. 27) show that Mab1 was capable of preventing or inhibiting of lungcancer tumor development in the murine xenograft model.

Example 19 Ability of Anti-B7-H3 Antibody to Prevent or Inhibit LOXMelanoma Tumor Development in a Murine Xenograft Model

Mice (eight female mCD16−/−, hCD16A+_FoxN1) were implantedsubcutaneously on their flanks with LOX-IMVI melanoma cancer cells andthen inoculated iv/Q7D×3 with PBS control, IgG control (5/mg/kg), Mab1(0.5, 1, 5 or 10 mg/kg), or ip/BIWx2 with Docetaxel (5, 10 or 20 mg/kg).The tumor cells were implanted in 200 μl of Ham's F12 Medium diluted 1:1with MATRIGEL™. Treatment with Mab1 was initiated within 7 days ofimplantation. The results of the experiment (FIGS. 28A-28C) show thatMab1 was capable of preventing or inhibiting of melanoma cancer tumordevelopment in the murine xenograft model.

Example 20 Ability of Anti-B7-H3 Antibody to Prevent or Inhibit UACC-62Melanoma Tumor Development in a Murine Xenograft Model

Mice (eight female mCD16−/−, hCD16A+_FoxN1) were implantedsubcutaneously on their flanks with UACC-62 melanoma cancer cells andthen inoculated iv/Q7D×5 with PBS control, IgG control (5/mg/kg) or Mab1(0.5, 1, 5 or 10 mg/kg). The tumor cells were implanted in 200 μl ofHam's F12 Medium diluted 1:1 with MATRIGEL™. Treatment with Mab1 wasinitiated within 7 days of implantation. The results of the experiment(FIG. 29) show that Mab1 was capable of preventing or inhibiting ofmelanoma cancer tumor development in the murine xenograft model.

Example 21 Ability of Anti-B7-H3 Antibody to Prevent or Inhibit 22rvProstate Tumor Development in a Murine Xenograft Model

Mice (mCD16−/−, hCD16A+_FoxN1) were implanted subcutaneously on theirflanks with 6×10⁶ 22rv prostate cancer cells and then inoculatediv/Q7D×4 with PBS control, IgG (10 mg/kg), Mab1 (0.5, 1, 5, or 10 mg/kg;Q7D×5) or trastuzumab (1.7 or 15 mg/kg). The tumor cells were implantedin 200 μl of Ham's F12 Medium diluted 1:1 with MATRIGEL™. Treatment withMab1 was initiated within 7 days of implantation. The results of theexperiment (FIGS. 30A-30C) show that Mab1 was capable of preventing orinhibiting of prostate cancer tumor development in the murine xenograftmodel.

Example 22 Ability of Anti-B7-H3 Antibody to Bind Renal Cancer Cells andPrevent or Inhibit Tumor Development in a Murine Xenograft Model

A498 renal cancer cells were incubated in the presence of hBRCA84D,chBRCA84D and hBRCA84 (0264 Fc) variant and the cytotoxic effect ofthese antibodies was determined. The results of this experiment areshown in FIG. 31, and indicate that all three of the antibodies werecytotoxic to A498 cells.

IHC analysis of the A498 xenograft tumor tissue was conducted usingbiotinylated BRCA84D antibody (20 μg/ml), BRCA69D (5 μg/ml) andanti-Her2 antibody (20 μg/ml). BRCA84D antibody was found to bind 20-40%of tumor tissue (weakly to moderately: + or ++); BRCA69D was found tobind 80-100% of tumor tissue (moderately to strongly: ++ or +++).BRCA84D antibody was found to weakly bind 40% of UMUC-3 tumor tissue(+); BRCA69D was found to moderately or strongly bind 70% of such tumortissue (++ or +++); anti-Her2 antibody was found to variably bind 20% ofsuch tumor tissue (+−+++). As controls, anti-Her2 antibody was found tobind SKBR-3 cells (+++) and BRCA84D and BRCA69D were found to be able tobind Hs 700T cells (+++)

Mice (mCD16−/−, hCD16A+_FoxN1) were implanted subcutaneously on theirflanks with 5×10⁶ A498 renal cancer cells. The tumor cells wereimplanted in 200 μl of Ham's F12 Medium diluted 1:1 with MATRIGEL™.Treatment with Mab1 was initiated within 7 days of implantation via ivQ7D×5 using doses of 0.1, 0.5, 1, 5, or 10 mg/kg. Centuximab (anti-EGRFantibody) was administered to a control group of mice at doses of 1, 7,or 15 mg/kg. Additional control mice were injected with vehicle or with10 mg/kg IgG control. The results of the experiment (FIG. 32) show thatMab1 was capable of preventing or inhibiting renalcancer tumordevelopment in the murine xenograft model.

Mice (mCD16−/−, hCD16A+_FoxN1) were alternatively implantedsubcutaneously on their flanks with 5×10⁶ 786-0 renal cancer cells. Thetumor cells were implanted in 200 μl of Ham's F12 Medium diluted 1:1with MATRIGEL™. Treatment with Mab1 was initiated within 7 days ofimplantation via iv Q7D×5 using doses of 0.1, 0.5, 1, 5, or 10 mg/kg.Centuximab (anti-EGRF antibody) was administered to a control group ofmice at doses of 1, 7, or 15 mg/kg. Additional control mice wereinjected with vehicle or with 10 mg/kg IgG control. The results of theexperiment (FIGS. 33A-33B) show that Mab1 was capable of preventing orinhibiting renal cancer tumor development in the murine xenograft model.

The activity of Mab1 was compared with that of paclitaxel, a mitoticinhibitor used in cancer chemotherapy. Groups of eight female mice(mCD16−/−, hCD16A+_FoxN1) were implanted subcutaneously on their flankswith 786-0 renal cancer cells and then provided with Mab1 via iv Q7D atdoses of 0.1, 0.5, 1, 5, or 10 mg/kg. Paclitaxel was administered to acontrol group of eight such mice at a dose of 2.5 mg/kg on study day 21,28, and 35. Additional control mice (seven female per group) wereinjected with vehicle or with 5 mg/kg IgG control. The results of theexperiment (FIG. 34) show that Mab1 was capable of preventing orinhibiting renal cancer tumor development in the murine xenograft model.

Example 23 Cynomolgous Monkey Toxicology Study

A cynomolgous monket toxicology study is conducted in order to assessacute toxicology profile after a single dose of Mab1, determine thepharmacokinetic profile for Mab1, establish a time vs. dose relationshipfor induction of cytokines associated with effector cell activation, andassess the effect of drug treatment on the level of circulatingleukocytes (e.g., NK and T-cells).

Such a study may be designed to involve four groups of 6 monkeys (3males and 3 females) and to extend 7 weeks from initial treatment tofinal necropsy. Group 1 would comprise a control group that wouldreceive only vehicle for weeks 1 and 2. Four members of Group 1 (twomales and two females) would be sacrificed at week 3. The remainingmembers of Group 1 would receive additional vehicle at week 3 and besacrificed for necropsy at week 7. Groups 2-4 are experimental groupsthat would receive vehicle at week 1, and B7-H3 antibody (1, 30, or 100mg/kg, respectively) at week 2. Four members of each Group (two malesand two females) would be sacrificed at week 3. The remaining members ofeach Group would receive additional vehicle at week 3 and be sacrificedfor necropsy at week 7.

All infusions are well tolerated and no mortality or significant changesin body weight, clinical signs or serum chemistry are observed.Dose-dependent reductions in circulating NK cells but not in circulatingB- and T-cells are observed.

The study provides verification of cynomolgus monkey as a relevanttoxicological species. When contacted with normal human tissue, antibodyBRCA84D showed various degrees of staining intensity in liver, pancreas,colon, lung and adrenal cortex. Liver staining was relatively restrictedto sinusoid lining cells (fibroblast and kupffer cells). Pancreasstaining was observed in collagen fiber mainly and a small percentage ofthe epithelium (acinar cells and/or intercalated duct cells). Colonstaining was relatively restricted in apical membrane of cryptepithelium and fibroblast in mucosa. Lung showed very weak and patchystaining in the epithelium. BRCA84D showed good cross-reactivity incynomolgus monkey tissues in comparison to the human tissue profile withthe exception of the lack of staining in the liver and pancreas, andpossible expression of B7-H3 in cynomolgus monkey pituitary cells.

All publications and patents mentioned in this specification are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in its entirety. While theinvention has been described in connection with specific embodimentsthereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

What is claimed is:
 1. A nucleic acid molecule comprising a sequenceencoding a polypeptide comprising: (A) a light chain having the aminoacid sequence of SEQ ID NO: 117; or (B) a heavy chain having the aminoacid sequence of SEQ ID NO:
 119. 2. The nucleic acid molecule of claim1, wherein the encoded polypeptide comprises the light chain having theamino acid sequence of SEQ ID NO:
 117. 3. The nucleic acid molecule ofclaim 1, wherein the encoded polypeptide comprises the heavy chainhaving the amino acid sequence of SEQ ID NO:
 119. 4. The nucleic acidmolecule of claim 3, wherein the encoded polypeptide further comprisesthe light chain having the amino acid sequence of SEQ ID NO:
 117. 5. Thenucleic acid molecule of claim 1, wherein said nucleic acid moleculecomprises the light chain sequence of SEQ ID NO:
 118. 6. The nucleicacid molecule of claim 1, wherein said nucleic acid molecule comprisesthe heavy chain sequence of SEQ ID NO:
 120. 7. The nucleic acid moleculeof claim 6, wherein said nucleic acid molecule further comprises thelight chain sequence of SEQ ID NO:
 118. 8. The nucleic acid molecule ofclaim 1, wherein the encoded polypeptide is a single chain antibody, amonoclonal antibody, or a diabody.
 9. A vector comprising the nucleicacid molecule of claim
 1. 10. The vector of claim 9, which is anexpression vector.
 11. A host cell comprising the expression vector ofclaim
 10. 12. A method of producing a substantially purifiedimmunoglobulin or an antigen-binding fragment thereof, comprising thesteps of: (A) growing a cell line transformed with the nucleic acid ofclaim 1 under conditions in which the immunoglobulin or antigen-bindingfragment thereof is expressed; and (B) harvesting the expressedimmunoglobulin or antigen-binding fragment thereof.